Surface waters are heavily used in food production worldwide. Several human pathogens can survive in these waters for long periods and disseminate to food production environments, contaminating our food supply.

Leafy greens are responsible for nearly half of the produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States and recent investigations have implicated agricultural water as a potential source. Current FDA detection protocols require extensive analysis time. We aimed to use Oxford Nanopore rapid sequencing kits for an in-field determination of agricultural water microbiome and possible detection and characterization of STECs strain(s) in these samples. We tested the performance of the nanopore rapid sequencing kit (RAD004) for fast microbiome determination using the well characterized ZymoBIOMICS mock microbial community and the number of reads for each identified species was present in the expected proportion. Rapid sequencing kit (LRK001 and RAD004) library preparation of DNA extracted from agricultural water resulted in poor nanopore sequencing reactions, with low output (0.3–1.7 M reads), a high proportion of failed reads (50–60%), and highly sheared DNA before and after a magnetic bead clean up. To improve performance, we prepared a DNA library with the ligation kit (LSK109), which includes multiple cleaning steps, reducing inherent inhibitors and producing a better outcome (2.2 M reads, 15% failed reads). No definitive presence of STEC could be confirmed in any of the sites. Approximately 100 reads from each site (0.02% of total reads) were identified as Escherichia coli, but the specific strain or their virulence genes could not be detected. Sites 9, 10, and 12 were found to be positive for STEC presence by microbiological techniques after enrichment. The rapid sequencing kits can be appropriate for genus or species level microbial identification, but we recommend the use of the ligation kit for increased sequencing depth and removal of contaminants in agricultural water. However, we were not able to identify any STEC strains in these nanopore microbiome samples, due to low initial concentrations. The results from this pilot study provide preliminary evidence that MinION sequencing of agricultural water using the ligation kit has the potential to be used for rapid microbiome determination in the field with optimal results for water quality surveillance.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 1, January 2019 Increasingly, routine surveillance and monitoring of foodborne pathogens using whole-genome sequencing is creating opportunities to study foodborne illness epidemiology beyond routine outbreak investigations and case–control studies. Using a global phylogeny of Salmonella enterica serotype Typhimurium, we found that major livestock sources of the pathogen in the United States can be predicted through whole-genome sequencing data. Relatively steady rates of sequence divergence in livestock lineages enabled the inference of their recent origins. Elevated accumulation of lineage-specific pseudogenes after divergence from generalist populations and possible metabolic acclimation in a representative swine isolate indicates possible emergence of host adaptation. We developed and retrospectively applied a machine learning Random Forest classifier for genomic source prediction of Salmonella Typhimurium that correctly attributed 7 of 8 major zoonotic outbreaks in the United States during 1998–2013. We further identified 50 key genetic features that were sufficient for robust livestock source prediction.

The GenomeTrakr database is a web-based tool utilized by a network of state and federal public health laboratories that collect and share genomic and geographic data from foodborne pathogens, including Salmonella enteritidis, Escherichia coli, Listeria monocytogenes, and Campylobacter jejuni. It consists of automated data storage and analysis capabilities housed in public databases at the National Center for Biotechnology Information (NCBI, Pathogen Detection). The information is accessible to researchers and public health officials for the purposes of quickly detecting foodborne disease outbreaks, and then taking the necessary steps to curtail further transmission of the pathogens The system is intended to speed foodborne illness outbreak detection and subsequent investigations that may ultimately reduce foodborne illnesses and deaths. The emerging network of public health laboratories using GenomeTrakr is using whole genome DNA sequencing as a typing tool for foodborne pathogens.

Salmonella is a leading cause of foodborne illness worldwide, and foods containing Salmonella (except raw meat and poultry products) are considered adulterated. Serotyping of Salmonella is an essential part of surveillance and investigation of outbreaks. This study evaluated a bead-based Salmonella molecular serotyping (SMS) method, which included the O-group 1, H-antigen, alternate target, and O-group 2 assays, compared with traditional serotyping. Salmonella was isolated from food, pet food, and environmental samples or were reference strains. A total of 572 isolates were analyzed by using two formats of the SMS method in comparison with traditional methods: 485 were analyzed by using Radix SMS (a custom user-mixed format), 218 were analyzed by using Luminex SMS (a commercial kit format), and 131 of the total isolates were analyzed by both formats for comparison. The SMS method was evaluated on the basis of the successful identification of antigens by the probes included in the method. The method identified 550 (96.2%) isolates as expected, 6 (1.0%) isolates were not identified as initially expected but were shown to be correctly identified by SMS after reanalysis by traditional serotyping, and 16 (2.8%) isolates not identified as expected possessed an antigen that should have been detected by the method but was not. Among the isolates considered correctly identified, 255 (44.6%) were identified to a single serovar, 44 (7.7%) required additional biochemical testing to differentiate variants or subspecies, and 251 (43.9%) were partially serotyped because probes for some antigens were not in the assay or had allelic variation for known serovars. Whole genome sequencing, SeqSero, and the Salmonella In Silico Typing Resource gave added confirmation for three isolates. Addition of the O-group 2 assay enabled the identification of 55 (9.6%) of 572 isolates. The SMS method could fully or partially serotype most isolates within a day. The SMS method should be a valuable tool when faster screening methods are needed, such as outbreaks and screening large numbers of environmental isolates.

AIMS:The effect of insect exclusion via netting on bacterial microbiota associated with field-grown tomato fruit and flowers was evaluated. METHODS AND RESULTS:Amplicon-based bacterial community profiling from insect-exposed plants and plants wrapped in nylon mosquito netting was conducted on total DNA extracted from tomato flower and mature unripe fruit washes. The V1-V3 region of the 16S rRNA gene was sequenced using Illumina MiSeq and analysed using qiime ver. 1.8. The carposphere supported significantly more phylogenetic diversity (PD) compared to the anthosphere, as measured by operational taxonomic unit richness (P = 0·001) and Faith's PD (P = 0·004). Flowers and fruit hosted distinct bacterial community structures (R2 = 0·27, P = 0·001), with specific taxonomic differences in taxa that included the Xanthomonadaceae (higher in flowers), and the Pseudomonadaceae, Methylobacteriaceae and Rhizobiales (higher in fruit) (FDR-P < 0·05). Bacterial community profiles of netted plants were overall statistically similar to non-netted plants for both flowers and fruit (P > 0·10). However, less variation between samples was observed among flowers (~50% less, P = 0·004) and green fruit (~10% less, P = 0·038) collected from netted than non-netted plants. CONCLUSION:Insects may introduce or augment variability in bacterial diversity associated with tomato flowers and potentially green fruit surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY:This work contributes to knowledge on microbiome dynamics of the tomato holobiont. Deciphering drivers of bacterial diversity and community structure of fruit crops could reveal processes important to agricultural management, such as competitive exclusion of pathogens and priming of plant defense mechanisms.

The incidence of food-borne illness outbreaks caused by contaminated fresh and fresh-cut vegetables has increased globally in recent years. This chapter presents a review of the microbial ecology of fresh and fresh-cut vegetables and their relationship to the major food-borne bacterial pathogens. Vegetables are known to harbor a diverse and complex array of bacterial communities. Numerous studies have examined the growth or die-off of pathogens under variable temperature conditions experienced during the processing, storage, and shipping of fresh-cut or minimally processed vegetables. During post-harvest processing, various routes of contamination with human pathogens may include contaminated water used for washing, chill tanks or sprays and shipping ice, processing equipment and transportation, infected workers, and cross-contamination from food preparation, display, and storage. It has become clear that natural selection plays a significant role in the adaptive change now observed among Salmonella strains associated with the food supply, produce notwithstanding.

Yi Chen, Yan Luo, Heather Carleton, Ruth Timme, David Melka, Tim Muruvanda, Charles 4 Wang, George Kastanis, Lee S. Katz, Lauren Turner, Angela Fritzinger, Terence Moore, 5 Robert Stones, Joseph Blankenship, Monique Salter, Mickey Parish, Thomas S. Hammack, 6 Peter S. Evans, Cheryl L. Tarr, Marc W. Allard, Errol A. Strain, Eric W. Brown 7 Food and Drug Administration, College Park, MD 20740 8 Centers for Disease Control and Prevention, Atlanta GA 30329 9 Virginia Division of Consolidated Laboratory Services, Richmond, VA 23219 10 Maryland Department of Health & Mental Hygiene, Baltimore, MD 21205 11 Robert Stones, Newcastle University, Newcastle upon Tyne, United Kingdom 12 13 *corresponding author ,yi.chen@fda.hhs.gov 14 Running title: whole genome and core genome sequencing analyses 15

Isothermal amplification assay is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of the 3M Molecular Detection System (MDS) and ANSR Pathogen Detection System (PDS) for the detection of Salmonella in egg products as compared to the Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method and a modified culture method (3M MDS and ANSR PDS preferred method). Two Salmonella ser. Enteritidis (18579, PT4; CDC_2010K_1441, PT8), one Salmonella ser. Heidelberg (607310-1), and one Salmonella ser. Typhimurium (0723) isolates were used in this study. Seven wet egg products and 13 dry egg products were inoculated with these strains individually at 1 to 5 CFU/25 g. One set of test portions was prepared following FDA BAM procedures [with lactose broth (LB) as pre-enrichment broth]. Another set of test portions was prepared using buffered peptone water (BPW) as pre-enrichment broth, as instructed by the 2 detection systems. Results from 3M MDS and ANSR PDS were 100% in agreement with their BPW-based culture method results. When LB was used as pre-enrichment broth, the number of Salmonella positive test portions (80 tested), identified with the BAM, 3M MDS, and ANSR PDS, were 63, 61, and 60, respectively. In conclusion, both 3M MDS and ANSR PDS Salmonella assays were as effective as their BPW based culture methods and were equivalent to the BAM culture method for the detection of Salmonella in egg products. These sensitive isothermal assays can be used as rapid detection tools for Salmonella in egg products provided that BPW is used as pre-enrichment broth.

This review is intended to help researchers generate hypotheses about chemicals that may contribute to diabetes and to obesity-related health outcomes by summarizing relevant findings from the U.S. Environmental Protection Agency (EPA) ToxCastTM high-throughput screening (HTS) program. The aim was to develop new hypotheses around environmental chemicals of potential interest for diabetesor obesity-related outcomes using high-throughput screening data. Researchers identified ToxCastTM assay targets relevant to several biological processes related to diabetes and obesity (insulin sensitivity in peripheral tissue, pancreatic islet and β cell function, adipocyte differentiation, and feeding behavior) and presented chemical screening data against those assay targets to identify chemicals of potential interest. The results of this screening-level analysis suggest that the spectrum of environmental chemicals to consider in research related to diabetes and obesity is much broader than indicated by research papers and reviews published in the peer-reviewed literature. Testing hypotheses based on ToxCastTM data will also help assess the predictive utility of this HTS platform. More research is required to put these screening-level analyses into context, but the information presented in this review should facilitate the development of new hypotheses.

Salmonella enterica commonly colonizes the intestinal tract of cattle and is a leading cause of foodborne illness. A previously described investigation into the prevalence of S. enterica on a dairy farm revealed an 8-year-long asymptomatic S. enterica epidemic caused by serotypes Cerro and Kentucky in the lactating herd. To investigate the source of the S. Kentucky strains, the genomes of two S. Kentucky isolates were sequenced; one collected prior to the epidemic (2004) and one collected during the epidemic (2010). Comparative genomic analysis demonstrated significant polymorphisms between the two strains. PCR primers targeting unique and strain-specific regions were developed, and screening of the archived isolates identified the index case of the asymptomatic S. Kentucky epidemic as a heifer that was raised off-site and transported onto the study farm in 2005. Analysis of isolates collected from all heifers brought onto the farm demonstrated frequent re-introduction of clones of the epidemic strain suggesting transmission of pathogens between farms might occur repeatedly.

Preprocessing results of 16S rRNA sequences. (XLSX 15 kb)

Although new serotypes of enterohemorrhagic Escherichia coli (EHEC) emerge constantly, the mechanisms by which these new pathogens arise and the reasons emerging serotypes tend to carry more virulence genes than other E. coli are not understood. An insertion sequence (IS) excision enhancer (IEE) was discovered in EHEC O157:H7 that promoted the excision of IS3 family members and generating various genomic deletions. One IS3 family member, IS629, actively transposes and proliferates in EHEC O157:H7 and enterotoxigenic E. coli (ETEC) O139 and O149. The simultaneous presence of the IEE and IS629 (and other IS3 family members) may be part of a system promoting not only adaptation and genome diversification in E. coli O157:H7 but also contributing to the development of pathogenicity among predominant serotypes. Prevalence comparisons of these elements in 461 strains, representing 72 different serotypes and 5 preassigned seropathotypes (SPT) A to E, showed that the presence of these two elements simultaneously was serotype specific and associated with highly pathogenic serotypes (O157 and top non-O157 Shiga toxin-producing Escherichia coli [STEC]) implicated in outbreaks and sporadic cases of human illness (SPT A and B). Serotypes lacking one or both elements were less likely to have been isolated from clinical cases. Our comparisons of IEE sequences showed sequence variations that could be divided into at least three clusters. Interestingly, the IEE sequences from O157 and the top 10 non-O157 STEC serotypes fell into clusters I and II, while less commonly isolated serotypes O5 and O174 fell into cluster III. These results suggest that IS629 and IEE elements may be acting synergistically to promote genome plasticity and genetic diversity among STEC strains, enhancing their abilities to adapt to hostile environments and rapidly take up virulence factors.

BACKGROUND Contamination of tomatoes by Salmonella can occur in agricultural settings. Little is currently understood about how agricultural inputs such as pesticide applications may impact epiphytic crop microflora and potentially play a role in contamination events. We examined the impact of two materials commonly used in Virginia tomato agriculture: acibenzolar-S-methyl (crop protectant) and copper oxychloride (pesticide) to identify the effects these materials may exert on baseline tomato microflora and on the incidence of three specific genera; Salmonella, Xanthomonas and Paenibacillus. RESULTS Approximately 186 441 16S rRNA gene and 39 381 18S rRNA gene sequences per independent replicate were used to analyze the impact of the pesticide applications on tomato microflora. An average of 3 346 677 (634 892 974 bases) shotgun sequences per replicate were used for metagenomic analyses. CONCLUSION A significant decrease in the presence of Gammaproteobacteria was observed between controls and copper-treated plants, suggesting that copper is effective at suppressing growth of certain taxa in this class. A higher mean abundance of Salmonella and Paenibacillus in control samples compared to treatments may suggest that both systemic and copper applications diminish the presence of these genera in the phyllosphere; however, owing to the lack of statistical significance, this could also be due to other factors. The most distinctive separation of shared membership was observed in shotgun data between the two different sampling time-points (not between treatments), potentially supporting the hypothesis that environmental pressures may exert more selective pressures on epiphytic microflora than do certain agricultural management practices. © 2014 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Salmonella enterica ssp. enterica serovar Enteritidis is the leading reported cause of Salmonella infections. Most Salmonella Enteritidis infections are associated with whole shell eggs and egg products. This project attempted to lay the foundation for improving the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella Enteritidis in shell eggs. Two Salmonella Enteritidis isolates were used for comparisons among different preenrichment and enrichment media and for the evaluation of egg:preenrichment broth ratios for the detection of Salmonella Enteritidis in shell eggs. The effect of surface disinfection on the detection of Salmonella Enteritidis in shell eggs was also investigated. The results indicated that tryptic soy broth (TSB) was similar to TSB plus ferrous sulfate, but significantly (α = 0.05) better than nutrient broth, Universal Preenrichment broth, and buffered peptone water when used for preenrichment of Salmonella in shell eggs. Salmonella Enteritidis populations after enrichment with Rappaport-Vassiliadis broth were 0.40 to 1.11 log cfu/mL of culture lower than those in preenrichment cultures. The reduction was statistically significant (α = 0.05). Egg:broth ratios at 1:9 and 1:2 produced significantly (α = 0.05) higher Salmonella Enteritidis populations after preenrichment with TSB with inoculum levels at 4 cfu/100 g of eggs and 40 cfu/1,000 g of eggs than the ratio at 1:1. Salmonella Enteritidis populations in TSB preenrichment cultures of shell eggs surface-disinfected with 70% alcohol:iodine/potassium iodide solution and untreated control were 9.11 ± 0.11 and 9.18 ± 0.05 log cfu/mL, respectively, for SE 13-2, and 9.20 ± 0.04 and 9.16 ± 0.05 log cfu/mL, respectively, for SE CDC_2010K_1543. Surface disinfection of eggs did not reduce the sensitivity of detection of Salmonella Enteritidis in liquid eggs. These results could improve the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella in shell eggs by simplifying the preenrichment medium and changing the sample handling before enrichment.

Aim: This report describes the use of a six-gene multi-locus sequence analysis (MLSA) to correctly identify Vibrio strains of the Harveyi clade. Methods and Results: Vibrio isolates were characterized using a six housekeeping gene MLSA. The study provided evidence supporting: (i) a substantial number of reference strains maintained within commercial culture collections are misidentified taxonomically at the species level; (ii) two V. alginolyticus subclades retain species-level divergence; and (iii) V. communis and V. owensii likely are the same species. Conclusion: A significant number (n = 10) of Harveyi clade Vibrio strains have been inaccurately identified, including evidence that V. communis and V. owensii strains, two recently discovered species assigned to the Harveyi clade, comprise a single species. Significance and Impact of the study: As Harveyi clade vibrios have an enormous impact on human and aquatic animal health, it is of paramount importance to identify members of the Harveyi clade correctly.

Listeria monocytogenes has caused numerous human outbreaks. Here we report draft genomes of L. monocytogenes J1816 and J1-220, which belong to epidemic clones II and IV, respectively. Whole-genome sequence analysis of these strains provides a tool for studying the short-term evolution of these epidemic clones.