Investigation of a Multistate Outbreak of Listeria monocytogenes Infections Linked to Frozen Vegetables Produced at Individually Quick-Frozen Vegetable Manufacturing Facilities.

In 2016, the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and state partners investigated nine Listeria monocytogenes infections linked to frozen vegetables. The investigation began with two environmental L. monocytogenes isolates recovered from Manufacturer A, primarily a processor of frozen onions, that were a match by whole genome sequencing (WGS) to eight clinical isolates and historical onion isolates with limited collection details. Epidemiologic information, product distribution, and laboratory evidence linked suspect food items, including products sourced from Manufacturer B, also a manufacturer of frozen vegetable/fruit products, with an additional illness. The environmental isolates were obtained during investigations at Manufacturers A and B. State and federal partners interviewed ill people, analyzed shopper card data, and collected household and retail samples. Nine ill persons between 2013 and 2016 were reported in four states. Of four ill people with information available, frozen vegetable consumption was reported by three, with shopper cards confirming purchases of Manufacturer B brands. Two identified outbreak strains of L. monocytogenes (Outbreak Strain 1 and Outbreak Strain 2) were a match to environmental isolates from Manufacturer A and/or isolates from frozen vegetables recovered from open and unopened product samples sourced from Manufacturer B; the investigation resulted in extensive voluntary recalls. The close genetic relationship between isolates helped investigators determine the source of the outbreak and take steps to protect public health. This is the first known multistate outbreak of listeriosis in the United States linked to frozen vegetables and highlights the significance of sampling and WGS analyses when there is limited epidemiologic information. Additionally, this investigation emphasizes the need for further research regarding food safety risks associated with frozen foods.

An Overview of Foodborne Sample-Initiated Retrospective Outbreak Investigations and Interagency Collaboration in the United States.

Foodborne outbreak investigations have traditionally included the detection of a cluster of illnesses first, followed by an epidemiologic investigation to identify a food of interest. The increasing use of whole genome sequencing (WGS) subtyping technology for clinical, environmental, and food isolates of foodborne pathogens, and the ability to share and compare the data on public platforms, present new opportunities to identify earlier links between illnesses and their potential sources. We describe a process called sample-initiated retrospective outbreak investigations (SIROIs) used by federal public health and regulatory partners in the United States. SIROIs begin with an evaluation of the genomic similarity between bacterial isolates recovered from food or environmental samples and clusters of clinical isolates while subsequent and parallel epidemiologic and traceback investigations are initiated to corroborate their connection. SIROIs allow for earlier hypothesis generation, followed by targeted collection of information about food exposures and the foods and manufacturer of interest, to confirm a link between the illnesses and their source. This often leads to earlier action that could reduce the breadth and burden of foodborne illness outbreaks. We describe two case studies of recent SIROIs and present the benefits and challenges. Benefits include insight into foodborne illness attribution, international collaboration, and opportunities for enhanced food safety efforts in the food industry. Challenges include resource intensiveness, variability of epidemiologic and traceback data, and an increasingly complex food supply chain. SIROIs are valuable in identifying connections among small numbers of illnesses that may span significant time periods; detecting early signals for larger outbreaks or food safety issues associated with manufacturers; improving our understanding of the scope of contamination of foods; and identifying novel pathogen/commodity pairs.

Multistate Outbreak Investigation of Salmonella Infections Linked to Kratom: A Focus on Traceback, Laboratory, and Regulatory Activities.

ABSTRACT: During spring 2018, the U.S. Food and Drug Administration (FDA), Centers for Disease Control and Prevention, and state and local public health agencies responded to a multistate outbreak of gastrointestinal illnesses caused by multiple Salmonella serovars and associated with consumption of kratom, a product harvested from a tropical tree native to Southeast Asia. The outbreak included 199 case-patients reported by 41 U.S. states, with illness onset dates ranging 11 from January 2017 to 8 May 2018, leading to 54 hospitalizations and no deaths. Case-patients reported purchasing kratom products from physical and online retail points of service (POSs). Products distributed to 16 POSs where 24 case-patients from 17 states purchased kratom were selected for traceback investigation. Traceback revealed that the kratom was imported from several countries, the most common being Indonesia. Local and state officials collected product samples from case-patients and retail POSs. The FDA collected 76 product samples from POSs and distributors, of which 42 (55%) tested positive for Salmonella. The positive samples exhibited a range of pulsed-field gel electrophoresis patterns and whole genome sequence genetic heterogeneity, and 25 (60%) of 42 samples yielded at least one isolate indistinguishable from one or more outbreak-related clinical isolates. Although it does not exclude a possibility of a single contamination source, the extent of genetic diversity exhibited by the Salmonella isolates recovered from product samples and a lack of traceback convergence suggested that kratom was widely contaminated across multiple sites from which it was grown, harvested, and packaged. As a result of the contamination, kratom products were recalled by numerous firms (both voluntarily and mandatory). Epidemiologic, traceback, and laboratory evidence supported the conclusion that kratom products were associated with illnesses.

Genomic evidence of environmental and resident Salmonella Senftenberg and Montevideo contamination in the pistachio supply-chain.

Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed an in-depth retrospective data analysis of Salmonella associated with pistachios as well as a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed utilizing short-read and long-read sequencing technologies. The sequence data were analyzed using two methods: the FDA's Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multilocus Sequence Typing (wgMLST). The year-long storage study evaluated the survival of five strains of Salmonella on pistachios stored at 25 °C at 35% and 54% relative humidity (RH). Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) the use of metagenomic analysis is a novel tool for determining the composition of serovar survival in a cocktail inoculated storage study.

A Series of Papaya-Associated Salmonella Illness Outbreak Investigations in 2017 and 2019: A Focus on Traceback, Laboratory, and Collaborative Efforts.

ABSTRACT: In 2017 and 2019, five outbreaks of infections from multiple strains of Salmonella linked to the consumption of whole, fresh Maradol papayas were reported in the United States, resulting in 325 ill persons. Traceback, laboratory, and epidemiologic evidence indicated papayas as the likely vehicle for each of these outbreaks and identified the source of papayas. State and U.S. Food and Drug Administration (FDA) laboratories recovered Salmonella from papaya samples from various points of distribution, including at import entry, and conducted serotyping, pulsed-field gel electrophoresis, and phylogenetic analyses of whole genome sequencing data. Federal and state partners led traceback investigations to determine the source of papayas. Four different suppliers of papayas were linked by traceback and laboratory results to five separate outbreaks of Salmonella infections associated with papayas. In 2017, multiple states tested papaya samples collected at retail, and Maryland and Virginia investigators recovered strains of Salmonella associated with one outbreak. FDA collected 183 papaya samples in 2017, and 11 samples yielded 62 isolates of Salmonella. Eleven serotypes of Salmonella were recovered from FDA papaya samples, and nine serotypes were closely related genetically by pulsed-field gel electrophoresis and whole genome sequencing to clinical isolates of four outbreaks, including the outbreak associated with positive state sample results. Four farms in Mexico were identified, and their names were released to the general public, retailers, and foreign authorities. In 2019, FDA collected 119 papaya samples, three of which yielded Salmonella; none yielded the 2019 outbreak strain. Investigators determined that papayas of interest had been sourced from a single farm in Campeche, Mexico, through traceback. This information was used to protect public health through public guidance, recalls, and import alerts and helped FDA collaborate with Mexican regulatory partners to enhance the food safety requirements for papayas imported from Mexico.

Lessons Learned from a Decade of Investigations of Shiga Toxin-Producing Escherichia coli Outbreaks Linked to Leafy Greens, United States and Canada.

Shiga toxin-producing Escherichia coli (STEC) cause substantial and costly illnesses. Leafy greens are the second most common source of foodborne STEC O157 outbreaks. We examined STEC outbreaks linked to leafy greens during 2009-2018 in the United States and Canada. We identified 40 outbreaks, 1,212 illnesses, 77 cases of hemolytic uremic syndrome, and 8 deaths. More outbreaks were linked to romaine lettuce (54%) than to any other type of leafy green. More outbreaks occurred in the fall (45%) and spring (28%) than in other seasons. Barriers in epidemiologic and traceback investigations complicated identification of the ultimate outbreak source. Research on the seasonality of leafy green outbreaks and vulnerability to STEC contamination and bacterial survival dynamics by leafy green type are warranted. Improvements in traceability of leafy greens are also needed. Federal and state health partners, researchers, the leafy green industry, and retailers can work together on interventions to reduce STEC contamination.

Frequency of Animal Leptospirosis in the Southern United States and the Implications for Human Health.

Leptospirosis is a zoonotic disease with symptoms in humans and animals, ranging from subclinical to serious and fatal. The disease occurs worldwide, but there is limited recognition of the public and animal health risks it poses in the southern United States. A systematic review of the frequency of animal leptospirosis in 17 states and jurisdictions covering the southern continental United States was performed to advance our understanding of the pathogen's distribution and identify transmission patterns that could be targeted for prevention efforts. Fifty-two articles, spanning >100 years, met the analysis criteria. A wide range of techniques were used to measure seroprevalence and isolate the bacteria. The assessment identified exposure to Leptospira spp and Leptospira spp infection among a diverse range of species, spanning 22 animal families within 14 states, suggesting that the pathogen is distributed throughout the southern region. Disease frequency trends were assessed among animals in various habitats (all habitats, nonwild habitats, and wild habitats). The frequency of Leptospira spp detection in animals in wild habitats increased slightly over time (<0.2%/year). We identified reports of 11 human leptospirosis illness clusters and outbreaks in the southern United States. Exposure to potentially contaminated surface waters were documented for at least seven of the events, and interactions with infected or likely infected animals were documented for at least six of the events. This analysis highlights the need for stronger partnerships across the public and animal health fields to enhance diagnostics, surveillance, and reporting. The early identification of leptospirosis in animals may serve as an indicator of environmental contamination and trigger prevention measures, such as vaccinating companion animals and livestock, use of potable water, and the wearing of waterproof protective clothing near water that may be contaminated.

Conditions at the time of inoculation influence survival of attenuated Escherichia coli O157:H7 on field-inoculated lettuce.

The impact of plant development, environmental conditions at the time of inoculation, and inoculum concentration on survival of attenuated BSL1 Escherichia coli O157:H7 strain ATCC 700728 on field-grown romaine lettuce was evaluated over 3 years. E. coli 700728 was inoculated onto 4- and 6-week-old romaine lettuce plants in the Salinas Valley, CA, at night or the next morning with either low (5 log) or high (7 log) cell numbers per plant to simulate a single aqueous contamination event. At night, when leaf wetness and humidity levels were high, E. coli cell numbers declined by 0.5 log CFU/plant over the first 8-10 h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2 h. However, similar numbers of E. coli were retrieved from lettuce plants at 2 and 7 days. E. coli cell numbers per plant were significantly lower (P < 0.05) 7 days after application onto 4-week-old compared to 6-week-old plants. E. coli 700728 could be recovered by plating or enrichment from a greater proportion of plants for longer times when inoculated at high compared with low initial concentrations and after inoculation of 6-week-old plants compared with 4-week-old plants, even at the low initial inoculum. A contamination event near harvest or when leaf wetness and humidity levels are high may enhance survivability, even when low numbers of E. coli are introduced.

Evaluation of the U.S. Food and Drug Administration validated method for detection of Cyclospora cayetanensis in high-risk fresh produce matrices and a method modification for a prepared dish.

The performance of the U.S. Food and Drug Administration (FDA) validated method for regulatory detection of Cyclospora cayetanensis in leafy greens and berries was evaluated in additional high-risk fresh produce items and in a dish prepared with these produce commodities. The method was robust and reproducible in basil, parsley, shredded carrots, shredded cabbage and carrot mix, and could detect as few as 5 oocysts in 25 g samples. Some differences in C. cayetanensis detection were found among the fresh produce analyzed. Significantly lower target gene copy numbers per reaction were obtained with shredded carrots, and shredded cabbage and carrot mix compared to leafy greens, which highlights the importance of evaluating the performance characteristics of validated methods in different food matrices. In the prepared dish, coleslaw with dressing, the method was optimized to detect 5 oocysts in a 25 g sample by using 1.0% Alconox® in the washing solution instead of 0.1% as originally described. These data are important to assess the prevalence of C. cayetanensis in different produce items and to support outbreak investigations.

Complete Genome Sequences of Four Salmonella enterica subsp. enterica Serovar Senftenberg and Montevideo Isolates Associated with a 2016 Multistate Outbreak in the United States.

A multistate outbreak of 11 Salmonella infections linked to pistachio nuts occurred in 2016. In this announcement, we report the complete genome sequences of four Salmonella enterica subsp. enterica serovar Senftenberg and S. enterica subsp. enterica serovar Montevideo isolates from pistachios collected during the 2016 outbreak investigation.

Multistate Outbreak of Salmonella Anatum Infections Linked to Imported Hot Peppers - United States, May-July 2016.

Foodborne salmonellosis causes an estimated 1 million illnesses and 400 deaths annually in the United States (1). Salmonella Anatum is one of the top 20 Salmonella serotypes in the United States. During 2013-2015 there were approximately 300-350 annual illnesses reported to PulseNet, the national molecular subtyping network for foodborne disease surveillance. In June 2016, PulseNet identified a cluster of 16 Salmonella Anatum infections with an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern from four states.* In April 2016, the same PFGE pattern had been uploaded to PulseNet from an isolate obtained from an Anaheim pepper, a mild to medium hot pepper. Hot peppers include many pepper varieties, such as Anaheim, jalapeño, poblano, and serrano, which can vary in heat level from mild to very hot depending on the variety and preparation. This rare PFGE pattern had been seen only 24 times previously in the PulseNet database, compared with common PFGE patterns for this serotype which have been seen in the database hundreds of times. Local and state health departments, CDC, and the Food and Drug Administration (FDA) investigated to determine the cause of the outbreak. Thirty-two patients in nine states were identified with illness onsets from May 6-July 9, 2016. Whole-genome sequencing (WGS) was performed to characterize clinical isolates and the Anaheim pepper isolate further. The combined evidence indicated that fresh hot peppers were the likely source of infection; however, a single pepper type or source farm was not identified. This outbreak highlights challenges in reconciling epidemiologic and WGS data, and the difficulties of identifying ingredient-level exposures through epidemiologic investigations alone.

Growth and survival of Enterobacteriaceae and inoculated Salmonella on walnut hulls and maturing walnut fruit.

Postharvest contamination of in-shell walnuts may occur when the fruit is dropped to or harvested from the orchard floor or as the outer hull is removed with mechanical abrasion and water. To evaluate the effect of maturity on the potential for microbial contamination, 'Howard' walnut fruits were collected weekly from the tree canopy, from 6 to 7 weeks before to 1 week after typical commercial harvest. The numbers of microorganisms able to form colonies on plate count agar, MacConkey agar (presumptive Enterobacteriaceae), or violet red bile lactose agar (presumptive coliforms) were compared on whole walnut fruits collected by hand directly from the tree or after exposure to the orchard floor for 10 min or 24 h. Salmonella Enteritidis PT 30 was inoculated at <1 to 8 log CFU/g onto 5-g hull pieces (from walnut fruit of different maturities) and stored at ambient temperature (23 to 26°C) in unsealed bags (38 to 90% relative humidity [RH] within bag) or in low humidity (20 to 45% RH) or high humidity (68 to 89% RH) for up to 14 days. Salmonella at 2 or 5 log CFU/ml was inoculated onto hulls before or up to 14 days after blending with water. As the walnut fruit matured, the indigenous bacterial levels on the surface increased, irrespective of whether fruit was collected from the tree or the ground. The RH influenced the growth of inoculated bacteria on hull pieces: Salmonella declined to <0.3 log CFU/g within 24 h at low RH but multiplied from 2 to 6 log CFU/g over 14 days of storage at >40% RH. Salmonella populations declined to <1 CFU/ml within 24 h in freshly blended green hulls but survived or multiplied in blended brown hulls or in blended green hulls that had been stored for 24 h or more before being inoculated.

Survival of foodborne pathogens on inshell walnuts.

The survival of Salmonella enterica Enteritidis PT 30 or five-strain cocktails of S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on inshell walnuts during storage. Inshell walnuts were separately inoculated with an aqueous preparation of the pathogens at levels of 10 to 4 log CFU/nut, dried for 24 h, and then stored at either 4 °C or ambient conditions (23-25 °C, 25-35% relative humidity) for 3 weeks to more than 1 year. During the initial 24-h drying period, bacterial levels declined by 0.7 to 2.4 log CFU/nut. After the inoculum dried, further declines of approximately 0.1 log CFU/nut per month of Salmonella Enteritidis PT 30 levels were observed on inshell walnuts stored at 4 °C; at ambient conditions the rates of decline ranged from 0.55 to 2.5 log CFU/nut per month. Rates of decline were generally greater during the first few weeks of storage, particularly at lower inoculum levels. The survival of the five-strain cocktails inoculated at very low levels (under 400 CFU/nut) was determined during storage at ambient conditions. The pathogens could be recovered by either enumeration or enrichment from most samples throughout the 3-month storage period; reductions in bacterial levels from the beginning to end of storage were 0.7, 0.2, and 2.3 log CFU/nut for Salmonella, E. coli O157:H7, and L. monocytogenes, respectively. For 6% of all nut samples (14 of 234 samples), pathogens were isolated from the second but not first 24-h enrichment, suggesting that bacterial cells were viable but not easily culturable. Salmonella-inoculated walnuts were exposed for 2 min to water or a 3% solution of sodium hypochlorite (to mimic commercial brightening) either 24 h or 7 days after inoculation; treated nuts were dried for 24h and held at ambient conditions. Salmonella levels were reduced by less than 0.5 log or 2.4 to 2.6 log CFU/nut on water- or chlorine- treated walnuts, respectively, regardless of postinoculation treatment time. Additional reductions of 2.6 and 2.1 log CFU/nut were observed for water- and chlorine-treated walnuts, respectively, after storage for 2 weeks at ambient conditions. Bacterial foodborne pathogens are capable of long-term survival on the surface of inshell walnuts even when initial levels are low.

A framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the application of untreated soil amendments of animal origin on land used to grow produce that may be consumed raw.

Application of manure or soil amendments of animal origin (untreated soil amendments; UTSAs) to agricultural land has been a long-standing practice to maintain or improve soil quality through addition of organic matter, nitrogen, and phosphorus. Much smaller quantities of these types of UTSAs are applied to land used for food crops than to land used for animal grain and forage. UTSAs can harbor zoonotic enteric pathogens that may survive for extended periods after application. Additional studies are needed to enhance our understanding of preharvest microbial food safety hazards and control measures pertaining to the application of UTSAs especially for land used to grow produce that may be consumed raw. This document is intended to provide an approach to study design and a framework for defining the scope and type of data required. This document also provides a tool for evaluating the strength of existing data and thus can aid the produce industry and regulatory authorities in identifying additional research needs. Ultimately, this framework provides a means by which researchers can increase consistency among and between studies and facilitates direct comparison of hazards and efficacy of controls applied to different regions, conditions, and practices.

A dry-inoculation method for nut kernels.

A dry-inoculation method for almonds and walnuts was developed to eliminate the need for the postinoculation drying required for wet-inoculation methods. The survival of Salmonella enterica Enteritidis PT 30 on wet- and dry-inoculated almond and walnut kernels stored under ambient conditions (average: 23 °C; 41 or 47% RH) was then compared over 14 weeks. For wet inoculation, an aqueous Salmonella preparation was added directly to almond or walnut kernels, which were then dried under ambient conditions (3 or 7 days, respectively) to initial nut moisture levels. For the dry inoculation, liquid inoculum was mixed with sterilized sand and dried for 24 h at 40 °C. The dried inoculated sand was mixed with kernels, and the sand was removed by shaking the mixture in a sterile sieve. Mixing procedures to optimize the bacterial transfer from sand to kernel were evaluated; in general, similar levels were achieved on walnuts (4.8-5.2 log CFU/g) and almonds (4.2-5.1 log CFU/g). The decline of Salmonella Enteritidis populations was similar during ambient storage (98 days) for both wet-and dry-inoculation methods for both almonds and walnuts. The dry-inoculation method mimics some of the suspected routes of contamination for tree nuts and may be appropriate for some postharvest challenge studies.

A framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the quality of agricultural water contacting fresh produce that may be consumed raw.

Agricultural water may contact fresh produce during irrigation and/or when crop protection sprays (e.g., cooling to prevent sunburn, frost protection, and agrochemical mixtures) are applied. This document provides a framework for designing research studies that would add to our understanding of preharvest microbial food safety hazards and control measures pertaining to agricultural water. Researchers will be able to use this document to design studies, to anticipate the scope and detail of data required, and to evaluate previously published work. This document should also be useful for evaluating the strength of existing data and thus should aid in identifying future research needs. Use of this document by the research community may lead to greater consistency or comparability than currently exists among research studies, which may ultimately facilitate direct comparison of hazards and efficacy of controls among different commodities, conditions, and practices.

Survival of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated walnut kernels during storage.

The survival of single strains or cocktails of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on walnut kernels. Kernels were separately inoculated with an aqueous preparation of the pathogens at 3 to 10 log CFU/g, dried for 7 days, and then stored at 23°C for 3 weeks to more than 1 year. A rapid decrease of 1 to greater than 4 log CFU/g was observed as the inoculum dried. In some cases, the time of storage at 23°C did not influence bacterial levels, and in other cases the calculated rates of decline for Salmonella (0.05 to 0.35 log CFU/g per month) and E. coli O157:H7 (0.21 to 0.86 log CFU/g per month) overlapped and were both lower than the range of calculated declines for L. monocytogenes (1.1 to 1.3 log CFU/g per month). In a separate study, kernels were inoculated with Salmonella Enteritidis PT 30 at 4.2 log CFU/g, dried (final level, 1.9 log CFU/g), and stored at -20, 4, and 23°C for 1 year. Salmonella Enteritidis PT 30 declined at a rate of 0.10 log CFU/g per month at 23°C; storage time did not significantly affect levels on kernels stored at -20 or 4°C. These results indicate the long-term viability of Salmonella, E. coli O157:H7, and L. monocytogenes on walnut kernels and support inclusion of these organisms in hazard assessments.

Fate of Escherichia coli O157:H7 in field-inoculated lettuce.

Impact of drip and overhead sprinkler irrigation on the persistence of attenuated Escherichia coli O157:H7 in the lettuce phyllosphere was investigated using a split-plot design in four field trials conducted in the Salinas Valley, California, between summer 2007 and fall 2009. Rifampicin-resistant attenuated E. coli O157:H7 ATCC 700728 (BLS1) was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old lettuce plant foliage with a spray bottle at a level of 7 log CFU ml⁻¹. When E. coli O157:H7 was inoculated onto 2-week-old plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation, respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7 declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks post-inoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on lettuce.