Evaluation of the Increased Genetic Resolution and Utility for Source Tracking of a Recently Developed Method for Genotyping Cyclospora cayetanensis.

Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.

Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water.

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 µg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

Development of a targeted amplicon sequencing method for genotyping Cyclospora cayetanensis from fresh produce and clinical samples with enhanced genomic resolution and sensitivity.

Outbreaks of cyclosporiasis, an enteric illness caused by the parasite Cyclospora cayetanensis, have been associated with consumption of various types of fresh produce. Although a method is in use for genotyping C. cayetanensis from clinical specimens, the very low abundance of C. cayetanensis in food and environmental samples presents a greater challenge. To complement epidemiological investigations, a molecular surveillance tool is needed for use in genetic linkage of food vehicles to cyclosporiasis illnesses, estimation of the scope of outbreaks or clusters of illness, and determination of geographical areas involved. We developed a targeted amplicon sequencing (TAS) assay that incorporates a further enrichment step to gain the requisite sensitivity for genotyping C. cayetanensis contaminating fresh produce samples. The TAS assay targets 52 loci, 49 of which are located in the nuclear genome, and encompasses 396 currently known SNP sites. The performance of the TAS assay was evaluated using lettuce, basil, cilantro, salad mix, and blackberries inoculated with C. cayetanensis oocysts. A minimum of 24 markers were haplotyped even at low contamination levels of 10 oocysts in 25 g leafy greens. The artificially contaminated fresh produce samples were included in a genetic distance analysis based on haplotype presence/absence with publicly available C. cayetanensis whole genome sequence assemblies. Oocysts from two different sources were used for inoculation, and samples receiving the same oocyst preparation clustered together, but separately from the other group, demonstrating the utility of the assay for genetically linking samples. Clinical fecal samples with low parasite loads were also successfully genotyped. This work represents a significant advance in the ability to genotype C. cayetanensis contaminating fresh produce along with greatly expanding the genomic diversity included for genetic clustering of clinical specimens.

Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States.

ABSTRACT: This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water.

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Closed bacterial genomes from whole genome sequencing play an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using 24 hour enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore's EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken2), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow provides proof of concept for faster detection and complete genomic characterization of STECs from a complex microbial sample compared to current reporting protocols and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

Survey of Salmonella in raw tree nuts at retail in the United States.

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [<0.01, 1.09]), 2.52% (95% CI [1.02, 5.12]), and 2.37% (95% CI [0.96, 4.83]), respectively. The rates of Salmonella isolation from major/big-chain supermarkets (1381), small-chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically (P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed-field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. PRACTICAL APPLICATION: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts.

Whole genome sequencing uses for foodborne contamination and compliance: Discovery of an emerging contamination event in an ice cream facility using whole genome sequencing.

We review how FDA surveillance identifies several ways that whole genome sequencing (WGS) improves actionable outcomes for public health and compliance in a case involving Listeria monocytogenes contamination in an ice cream facility. In late August 2017 FDA conducted environmental sampling inside an ice cream facility. These isolates were sequenced and deposited into the GenomeTrakr databases. In September 2018 the Centers for Disease Control and Prevention contacted the Florida Department of Health after finding that the pathogen analyses of three clinical cases of listeriosis (two in 2013, one in 2018) were highly related to the aforementioned L. monocytogenes isolates collected from the ice cream facility. in 2017. FDA returned to the ice cream facility in late September 2018 and conducted further environmental sampling and again recovered L. monocytogenes from environmental subsamples that were genetically related to the clinical cases. A voluntary recall was issued to include all ice cream manufactured from August 2017 to October 2018. Subsequently, FDA suspended this food facility's registration. WGS results for L. monocytogenes found in the facility and from clinical samples clustered together by 0-31 single nucleotide polymorphisms (SNPs). The FDA worked together with the Centers for Disease Control and Prevention, as well as the Florida Department of Health, and the Florida Department of Agriculture and Consumer Services to recall all ice cream products produced by this facility. Our data suggests that when available isolates from food facility inspections are subject to whole genome sequencing and the subsequent sequence data point to linkages between these strains and recent clinical isolates (i.e., <20 nucleotide differences), compliance officials should take regulatory actions early to prevent further potential illness. The utility of WGS for applications related to enforcement of FDA compliance programs in the context of foodborne pathogens is reviewed.

Survey of Foodborne Pathogens, Aerobic Plate Counts, Total Coliform Counts, and Escherichia coli Counts in Leafy Greens, Sprouts, and Melons Marketed in the United States.

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.

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.

Prevalence of Salmonella in Cashews, Hazelnuts, Macadamia Nuts, Pecans, Pine Nuts, and Walnuts in the United States.

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.

Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks. Using WGS can delimit contamination sources for foodborne illnesses across multiple outbreaks and reveal otherwise undetected DNA sequence differences essential to the tracing of bacterial pathogens as they emerge.

Practical Value of Food Pathogen Traceability through Building a Whole-Genome Sequencing Network and Database.

The FDA has created a United States-based open-source whole-genome sequencing network of state, federal, international, and commercial partners. The GenomeTrakr network represents a first-of-its-kind distributed genomic food shield for characterizing and tracing foodborne outbreak pathogens back to their sources. The GenomeTrakr network is leading investigations of outbreaks of foodborne illnesses and compliance actions with more accurate and rapid recalls of contaminated foods as well as more effective monitoring of preventive controls for food manufacturing environments. An expanded network would serve to provide an international rapid surveillance system for pathogen traceback, which is critical to support an effective public health response to bacterial outbreaks.

Platform for identification of Salmonella serovar differentiating bacterial proteins by top-down mass spectrometry: S. Typhimurium vs S. Heidelberg.

Intact protein expression profiling has proven to be a powerful tool for bacterial subspecies differentiation. To facilitate typing, epidemiology, and trace-back of Salmonella contamination in the food supply, a minimum of serovar level differentiation is required. Subsequent identification and validation of marker proteins is integral to rapid screening development and to determining which proteins are subject to environmental pressure. Bacterial sequencing efforts have expanded the number of sequenced genomes available for single-nucleotide polymorphism (SNP) analyses, but annotation is often missing, start site errors are not uncommon, and the likelihood of expression is not known. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed serovar specific nonsynonymous SNPs. Combinations of these marker proteins can be used in assays for rapid differentiation of bacteria. LC-MS generated intact protein expression profiles establish which bacterial protein masses differ across samples and can be determined without prior knowledge of the sample. Subsequent top-down LC-MS/MS is used to identify expressed proteins and their post-translational modifications (PTM), identify serovar specific markers, and validate genomic predicted orthologues as expressed biomarkers.

The evolutionary history and diagnostic utility of the CRISPR-Cas system within Salmonella enterica ssp. enterica.

Evolutionary studies of clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (cas) genes can provide insights into host-pathogen co-evolutionary dynamics and the frequency at which different genomic events (e.g., horizontal vs. vertical transmission) occur. Within this study, we used whole genome sequence (WGS) data to determine the evolutionary history and genetic diversity of CRISPR loci and cas genes among a diverse set of 427 Salmonella enterica ssp. enterica isolates representing 64 different serovars. We also evaluated the performance of CRISPR loci for typing when compared to whole genome and multilocus sequence typing (MLST) approaches. We found that there was high diversity in array length within both CRISPR1 (median = 22; min = 3; max = 79) and CRISPR2 (median = 27; min = 2; max = 221). There was also much diversity within serovars (e.g., arrays differed by as many as 50 repeat-spacer units among Salmonella ser. Senftenberg isolates). Interestingly, we found that there are two general cas gene profiles that do not track phylogenetic relationships, which suggests that non-vertical transmission events have occurred frequently throughout the evolutionary history of the sampled isolates. There is also considerable variation among the ranges of pairwise distances estimated within each cas gene, which may be indicative of the strength of natural selection acting on those genes. We developed a novel clustering approach based on CRISPR spacer content, but found that typing based on CRISPRs was less accurate than the MLST-based alternative; typing based on WGS data was the most accurate. Notwithstanding cost and accessibility, we anticipate that draft genome sequencing, due to its greater discriminatory power, will eventually become routine for traceback investigations.

Phylogenomic analysis identifies gene gains that define Salmonella enterica subspecies I.

Comparative methods for analyzing whole genome sequence (WGS) data enable us to assess the genetic information available for reconstructing the evolutionary history of pathogens. We used the comparative approach to determine diagnostic genes for Salmonella enterica subspecies I. S. enterica subsp. I strains are known to infect warm-blooded organisms regularly while its close relatives tend to infect only cold-blooded organisms. We found 71 genes gained by the common ancestor of Salmonella enterica subspecies I and not subsequently lost by any member of this subspecies sequenced to date. These genes included many putative functional phenotypes. Twenty-seven of these genes are found only in Salmonella enterica subspecies I; we designed primers to test these genes for use as diagnostic sequence targets and data mined the NCBI Sequence Read Archive (SRA) database for draft genomes which carried these genes. We found that the sequence specificity and variability of these amplicons can be used to detect and discriminate among 317 different serovars and strains of Salmonella enterica subspecies I.

Phylogenetic diversity of the enteric pathogen Salmonella enterica subsp. enterica inferred from genome-wide reference-free SNP characters.

The enteric pathogen Salmonella enterica is one of the leading causes of foodborne illness in the world. The species is extremely diverse, containing more than 2,500 named serovars that are designated for their unique antigen characters and pathogenicity profiles-some are known to be virulent pathogens, while others are not. Questions regarding the evolution of pathogenicity, significance of antigen characters, diversity of clustered regularly interspaced short palindromic repeat (CRISPR) loci, among others, will remain elusive until a strong evolutionary framework is established. We present the first large-scale S. enterica subsp. enterica phylogeny inferred from a new reference-free k-mer approach of gathering single nucleotide polymorphisms (SNPs) from whole genomes. The phylogeny of 156 isolates representing 78 serovars (102 were newly sequenced) reveals two major lineages, each with many strongly supported sublineages. One of these lineages is the S. Typhi group; well nested within the phylogeny. Lineage-through-time analyses suggest there have been two instances of accelerated rates of diversification within the subspecies. We also found that antigen characters and CRISPR loci reveal different evolutionary patterns than that of the phylogeny, suggesting that a horizontal gene transfer or possibly a shared environmental acquisition might have influenced the present character distribution. Our study also shows the ability to extract reference-free SNPs from a large set of genomes and then to use these SNPs for phylogenetic reconstruction. This automated, annotation-free approach is an important step forward for bacterial disease tracking and in efficiently elucidating the evolutionary history of highly clonal organisms.

Co-enriching microflora associated with culture based methods to detect Salmonella from tomato phyllosphere.

The ability to detect a specific organism from a complex environment is vitally important to many fields of public health, including food safety. For example, tomatoes have been implicated numerous times as vehicles of foodborne outbreaks due to strains of Salmonella but few studies have ever recovered Salmonella from a tomato phyllosphere environment. Precision of culturing techniques that target agents associated with outbreaks depend on numerous factors. One important factor to better understand is which species co-enrich during enrichment procedures and how microbial dynamics may impede or enhance detection of target pathogens. We used a shotgun sequence approach to describe taxa associated with samples pre-enrichment and throughout the enrichment steps of the Bacteriological Analytical Manual's (BAM) protocol for detection of Salmonella from environmental tomato samples. Recent work has shown that during efforts to enrich Salmonella (Proteobacteria) from tomato field samples, Firmicute genera are also co-enriched and at least one co-enriching Firmicute genus (Paenibacillus sp.) can inhibit and even kills strains of Salmonella. Here we provide a baseline description of microflora that co-culture during detection efforts and the utility of a bioinformatic approach to detect specific taxa from metagenomic sequence data. We observed that uncultured samples clustered together with distinct taxonomic profiles relative to the three cultured treatments (Universal Pre-enrichment broth (UPB), Tetrathionate (TT), and Rappaport-Vassiliadis (RV)). There was little consistency among samples exposed to the same culturing medias, suggesting significant microbial differences in starting matrices or stochasticity associated with enrichment processes. Interestingly, Paenibacillus sp. (Salmonella inhibitor) was significantly enriched from uncultured to cultured (UPB) samples. Also of interest was the sequence based identification of a number of sequences as Salmonella despite indication by all media, that samples were culture negative for Salmonella. Our results substantiate the nascent utility of metagenomic methods to improve both biological and bioinformatic pathogen detection methods.