The effective control of foodborne salmonellosis relies on the rapid and reliable detection and identification of the pathogen. Reliable detection tools for identifying the most common Salmonella serovars should translate to a considerable alleviation of the health burden attributed to Salmonella. We have developed a PCR assay for the rapid identification of colonies of Salmonella enterica serovar Thompson, a common serovar. Genomic analyses of publicly available sequences of Salmonella Thompson revealed the presence of a unique, Thompson-specific fragment, which we have used to design a pair of oligonucleotides, STho-F and STho-R, for the PCR amplification of an 808 bp DNA fragment. Using crude DNA extracts, the 808 bp fragment was detected in 77 out of 78 isolates of S. Thompson (sensitivity = 98.7% n = 78 isolates) but not in any of the non-Salmonella organisms tested (n = 100; 100% specificity) nor in non-Thompson Salmonella serovars (n = 100; 100% specificity). The sensitivity (inclusivity) and specificity (exclusivity) indices of the PCR assay for S. Thompson met the standard regulatory requirements. The Thompson primer pair was compatible with other primers pairs in a multiplex PCR designed for three other common Salmonella serovars. Colonies belonging to the Enteritidis serovar (n = 100), Heidelberg serovar (n = 100), Typhimurium serovar (n = 100), and Thompson serovar (n = 77) were correctly designated, indicating excellent inclusivity and exclusivity scores for all four Salmonella serovars tested in a single multiplex PCR.

Iron is an essential mineral for almost all pathogenic bacteria, including Salmonella enterica serovar Typhimurium and Enteritidis. We have investigated the effect of the deletion of iroN, fepA, and fhu genes on the transcriptomic profiles of S. Typhimurium and S. Enteritidis strains with double (ΔiroNΔfepA) and triple (ΔfhuΔiroNΔfepA) gene deletions and grown under iron-deficient conditions. Significant changes were observed in the expression of genes involved in virulence, stress-response, and energy metabolism in both Salmonella serovars. The pathways most affected were the tricarboxylic acid cycle, electron transport chain, and stress responses all of which were downregulated while cysteine biosynthesis was upregulated. In general, common and/or related genes were affected in the double and triple mutant strains which indicated that the additional deletion of the fhu gene affected a limited number of genes, although S. Typhimurium showed more differences than S. Enteritidis. Nevertheless, the main difference observed between the mutants of the two serovars grown under iron-depleted conditions is the enhanced upregulation of the flagellum genes in S. Enteritidis. This study reveals evidence of novel interactions between metabolic pathways and virulence under iron limitation, providing insights into adaptive strategies that may contribute to the enhanced virulence of these Salmonella serovars.

Introduction:Infections with protozoan parasites associated with the consumption of fresh produce is an on-going issue in developed countries but mitigating the risk is hampered by the lack of adequate methods for their detection and identification. Materials and methods:We developed a metagenomic next-generation sequencing (mNGS) assay using a MinION sequencer for the identification of parasites in intentionally contaminated lettuce to achieve a more accurate and rapid method than the traditional molecular and microscopy methods commonly used for regulatory purposes. Lettuce (25 g) was spiked with varying numbers of Cryptosporidium parvum oocysts, and microbes washed from the surface of the lettuce were lysed using the OmniLyse device. DNA was then extracted by acetate precipitation, followed by whole genome amplification. The amplified DNA was sequenced by nanopore technology and validated with the Ion Gene Studio S5, and the generated fastq files raw reads were uploaded to the CosmosID webserver for the bioinformatic identification of microbes in the metagenome. To demonstrate the ability of the procedure to distinguish other common food and waterborne protozoan parasites, lettuce was also spiked with C. hominis, C. muris, Giardia duodenalis and Toxoplasma gondii individually or together. Results:The efficient lysis of oocysts and cysts was a prerequisite for the sensitive detection of parasite DNA and was rapidly achieved within 3 min. Amplification of extracted DNA led to the generation of 0.16-8.25 μg of DNA (median = 4.10 μg), sufficient to perform mNGS. Nanopore sequencing followed by bioinformatic analysis led to the consistent identification of as few as 100 oocysts of C. parvum in 25 g of fresh lettuce. Similar results were obtained using the Ion S5 sequencing platform. The assay proved useful for the simultaneous detection of C. parvum, C. hominis, C. muris, G. duodenalis and T. gondii. Discussion:Our metagenomic procedure led to the identification of C. parvum present on lettuce at low numbers and successfully identified and differentiated other protozoa either of the same genus or of different genera. This novel mNGS assay has the potential for application as a single universal test for the detection of foodborne parasites, and the subtyping of parasites for foodborne outbreak investigations and surveillance studies.

Salmonella causes significant illness in humans and animals and is a major public health concern worldwide, contributing to an increased economic burden. Salmonella is usually transmitted through the consumption of contaminated food, such as raw or undercooked meat, poultry, eggs, and dairy products, and water or through contact with infected animals or their environment. The most common symptoms of salmonellosis, the illness caused by Salmonella, include diarrhea, fever, and abdominal cramps; in severe cases, the infection can lead to hospitalization and even death. The classification and taxonomy of Salmonella were historically controversial, but the genus is now widely accepted as composed of two species and over 2600 serovars. Some of these serovars infect a single host, that is, host-restricted, whereas others have a broad host range. Colonization of the host is complex and involves a series of interactions between the Salmonella and the host’s immune system. Salmonella utilizes an array of over 300 virulence factors, mostly present in Salmonella pathogenicity islands (SPIs) to achieve adherence, invasion, immune evasion, and, occasionally, systemic infection. Once colonized, it secretes a number of toxins and inflammatory mediators that cause diarrhea and other symptoms of salmonellosis. The overuse and misuse of antibiotics in human and animal medicine and agriculture have contributed to the development of antimicrobial resistance (AMR) in Salmonella, making AMR strains more severe and difficult to treat and increasing the risk of morbidity and mortality. Various methods are used for the detection of Salmonella, including traditional culture methods, molecular methods such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), and immunological-based assays. Because of its ubiquitous distribution, the prevention and control of Salmonella transmission remain a significant challenge. This chapter briefly covers the history, classification, transmission, pathogenesis and virulence factors, antimicrobial resistance genes, detection, diagnosis, surveillance, prevention, and control pertaining to Salmonella.

The genome sequence of multidrug-resistant Raoultella terrigena RT01-5M1 strain isolated from Canadian farmed salmon was determined using Oxford nanopore and Illumina MiSeq sequencers. The assembled chromosome was estimated at 5,699,993 bp in size, with two plasmids, 164,879 bp and 82,046 bp. The chromosome and smaller plasmid contained antimicrobial resistance genes.

Glyphosate-resistant wheat plants were discovered in southern Alberta in 2017, representing an unauthorized GM release in Canada. The Canadian Food Inspection Agency undertook a series of experiments to characterize and identify this unknown GM wheat, as well as to develop and validate construct-specific and event-specific qPCR assays. Results of PCR-based assays and Sanger sequencing indicated the presence of CaMV 35S promoter (p35S), Rice Actin 1 intron (RactInt1), CP4-EPSPS gene and nopaline synthase terminator (tNOS) elements in the unknown GM wheat. Genome walking and bead capture strategies, combined with high-throughput sequencing, were used to identify the 5 ' and 3 ' wheat junctions and the subsequent mapping of the insert to chromosome 3B of the wheat genome. A probable transformation vector, pMON25497, was recognized, and further testing identified the unknown GM wheat as MON71200 event, one of two events obtained with the pMON25497 vector. The two construct-specific assays targeted the junctions of the RactInt1 and the CP4-EPSPS elements and the CP4-EPSPS and tNOS elements, while the event-specific assay was located at the 3 ' junction into the wheat genome. Both construct-specific and event-specific assays had limits of detection of 0.10% of MON71200 in a seed pool. As expected, the two construct-specific assays cross-reacted with other wheat and corn events containing the same elements in the same order. No cross-reactivity was observed for the event-specific assay. The integrated strategy employed in this study can serve as a model for other cases when facing similar challenges involving unknown GM events.

IntroductionOutbreak investigation of foodborne salmonellosis is hindered when the food source is contaminated by multiple strains of Salmonella, creating difficulties matching an incriminated organism recovered from patients with the specific strain in the suspect food. An outbreak of the rare Salmonella Adjame was caused by multiple strains of the organism as revealed by single-nucleotide polymorphism (SNP) variation. The use of highly discriminatory prophage analysis to characterize strains of Salmonella should enable a more precise strain characterization and aid the investigation of foodborne salmonellosis.MethodsWe have carried out genomic analysis of S. Adjame strains recovered during the course of a recent outbreak and compared them with other strains of the organism (n = 38 strains), using SNPs to evaluate strain differences present in the core genome, and prophage sequence typing (PST) to evaluate the accessory genome. Phylogenetic analyses were performed using both total prophage content and conserved prophages.ResultsThe PST analysis of the S. Adjame isolates showed a high degree of strain heterogeneity. We observed small clusters made up of 2-6 isolates (n = 27) and singletons (n = 11) in stark contrast with the three clusters observed by SNP analysis. In total, we detected 24 prophages of which only four were highly prevalent, namely: Entero_p88 (36/38 strains), Salmon_SEN34 (35/38 strains), Burkho_phiE255 (33/38 strains) and Edward_GF (28/38 strains). Despite the marked strain diversity seen with prophage analysis, the distribution of the four most common prophages matched the clustering observed using core genome.DiscussionMutations in the core and accessory genomes of S. Adjame have shed light on the evolutionary relationships among the Adjame strains and demonstrated a convergence of the variations observed in both fractions of the genome. We conclude that core and accessory genomes analyses should be adopted in foodborne bacteria outbreak investigations to provide a more accurate strain description and facilitate reliable matching of isolates from patients and incriminated food sources. The outcomes should translate to a better understanding of the microbial population structure and an 46 improved source attribution in foodborne illnesses.

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes, foodborne pathogen. This study conducted in silico genomic analysis to investigate the specificity and binding efficacy of four published pairs of PCR primers targeting Listeria prfA-virulence gene cluster (pVGC) based on Listeria sequences available. We first performed comprehensive genomic analyses of the pVGC, the main pathogenicity island in Listeria spp. In total, 2961 prfA, 642 plcB, 629 mpl, and 1181 hlyA gene sequences were retrieved from the NCBI database. Multiple sequence alignments and phylogenetic trees were generated using unique (non-identical or not-shared) sequences of each represented genes, targeting four pairs of PCR primers published previously, namely 202 prfA, 82 plcB, 150 mpl, and 176 hlyA unique gene sequences. Only the hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB, and mpl genes showed weak (<50%) matching results. In addition, nucleotide variations were observed at the 3 ' end of the primers, indicating non-binding to the targets could potentially cause false-negative results. Thus, we propose designing degenerate primers or multiple PCR primers based on as many isolates as possible to minimize the false-negative risk and reach the aim of low tolerable limits of detection.

We have developed a targeted, amplicon-based next-generation sequencing method to detect and analyze 227 virulence genes (VG) of Salmonella (AmpliSeqSalm_227VG) for assessing the pathogenicity potential of Salmonella. The procedure was developed using 80 reference genomes representing 75 epidemiologically-relevant serovars associated with human salmonellosis. We applied the AmpliSeqSalm_227VG assay to (a) 35 previously characterized field strains of Salmonella consisting of serovars commonly incriminated in foodborne illnesses and (b) 34 Salmonella strains with undisclosed serological or virulence attributes, and were able to divide Salmonella VGs into two groups: core VGs and variable VGs. The commonest serovars causing foodborne illnesses such as Enteritidis, Typhimurium, Heidelberg and Newport had a high number of VGs (217–227). In contrast, serovars of subspecies not commonly associated with human illnesses, such as houtenae, arizonae and salame, tended to have fewer VGs (177–195). Variable VGs were not only infrequent but, when present, displayed considerable sequence variation: safC, sseL, sseD, sseE, ssaK and stdB showed the highest variation and were linked to strain pathogenicity. In a chicken infection model, VGs belonging to rfb and sse operons showed differences and were linked with pathogenicity. The high-throughput, targeted NGS-based AmpliSeqSalm_227VG procedure provided previously unknown information about variation in select virulence genes that can now be applied to a much larger population of Salmonella for evaluating pathogenicity of various serovars of Salmonella and for risk assessment of foodborne salmonellosis.

Dissemination of microorganisms with antimicrobial resistance genes (ARGs) through the food chain has been recognized as a growing public health concern worldwide. Biosolids, a product of wastewater treatment process, have been used as fertilizers in agriculture globally and have also been considered as a potential source of pathogens and ARGs for horizontal transfer across various environments. This study characterized antibiotic resistomes and microbiota in 24 biosolids samples collected from two Canadian waste water treatment plants in different cities in 2009 and 2019. The ARGs were detected using a qPCR array kit, and microbiota was analyzed using 16S ribosomal RNA gene amplicon sequencing. Furthermore, correlation analysis of ARG abundance and bacterial genera abundance was explored to predict potential hosts of ARGs. Seventy-one of 84 ARGs were detected in at least one or more samples with 12 ARGs being detected in all samples. Antibiotic resistomes did not show a statistically significant distinction between different collection years, sites, or year and site combined in principle coordinate analysis. The microbiota communities were significantly different between samples collected in different years, sites, or year and site combined. In total 34 phyla were detected with 13 genera among the top three phyla were typically related to the human gut microbiota and seven of them showing strong correlation with ARGs related to aminoglycoside and beta-lactam resistance. This study provides valuable baseline information and consistent trend on ARGs and bacterial communities in biosolid fertilizers in Canada, indicating that the biosolid fertilizer could potentially be a source of ARGs in the agricultural soils and may leading to potential contamination of plant-based food chains.

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.

Salmonella enterica serovar Enteritidis (or Salmonella Enteritidis, SE) is one of the oldest members of the genus Salmonella, based on the date of first description and has only gained prominence as a significant bacterial contaminant of food over the last three or four decades. Currently, SE is the most common Salmonella serovar causing foodborne illnesses. Control measures to alleviate human infections require that food isolates be characterized and this was until recently carried out using Pulsed-Field Gel Electrophoresis (PFGE) and phage typing as the main laboratory subtyping tools for use in demonstrating relatedness of isolates recovered from infected humans and the food source. The results provided by these analytical tools were presented with easy-to-understand and comprehensible nomenclature, however, the techniques were inherently poorly discriminatory, which is attributable to the clonality of SE. The tools have now given way to whole genome sequencing which provides a full and comprehensive genetic attributes of an organism and a very attractive and superior tool for defining an isolate and for inferring genetic relatedness among isolates. A comparative phylogenomic analysis of isolates of choice provides both a visual appreciation of relatedness as well as quantifiable estimates of genetic distance. Despite the considerable information provided by whole genome analysis and development of a phylogenetic tree, the approach does not lend itself to generating a useful nomenclature-based description of SE subtypes. To this end, a highly discriminatory, cost-effective, high throughput, validated single nucleotide based genotypic polymerase chain reaction assay (SNP-PCR) was developed focussing on 60 polymorphic loci. The procedure was used to identify 25 circulating clades of SE, the largest number so far described for this organism. The new subtyping test, which exploited whole genome sequencing data, displays the attributes of an ideal subtyping test: high discrimination, low cost, rapid, highly reproducible and epidemiological concordance. The procedure is useful for identifying the subtype designation of an isolate, for defining the population structure of the organism as well as for surveillance and outbreak detection.

Salmonella Infantis, a common contaminant of poultry products, is known to harbor mobile genetic elements that confer multi-drug resistance (MDR) and have been detected in many continents. Here, we report four MDR S. Infantis strains recovered from poultry house environments in Santa Cruz Island of the Galapagos showing extended-spectrum β-lactamase (ESBL) resistance and reduced fluoroquinolone susceptibility. Whole-genome sequencing (WGS) revealed the presence of the ESBL-conferring blaCTX-M-65 gene in an IncFIB-like plasmid in three S. Infantis isolates. Multi-locus sequence typing (MLST) and single nucleotide variant/polymorphism (SNP) SNVPhyl analysis showed that the S. Infantis isolates belong to sequence type ST32, likely share a common ancestor, and are closely related (1–3 SNP difference) to blaCTX-M-65-containing clinical and veterinary S. Infantis isolates from the United States and Latin America. Furthermore, phylogenetic analysis of SNPs following core-genome alignment (i.e., ParSNP) inferred close relatedness between the S. Infantis isolates from Galapagos and the United States. Prophage typing confirmed the close relationship among the Galapagos S. Infantis and was useful in distinguishing them from the United States isolates. This is the first report of MDR blaCTX-M-65-containing S. Infantis in the Galapagos Islands and highlights the need for increased monitoring and surveillance programs to determine prevalence, sources, and reservoirs of MDR pathogens.

Background: Bacteriophages are bacterial parasites and are considered the most abundant and diverse biological entities on the planet. Previously we identified 154 prophages from 151 serovars of Salmonella enterica subsp. enterica . A detailed analysis of Salmonella prophage genomics is required given the influence of phages on their bacterial hosts and should provide a broader understanding of Salmonella biology and virulence and contribute to the practical applications of phages as vectors and antibacterial agents. Results: Comparative analysis of the full genome sequences of 142 prophages of Salmonella enterica subsp. enterica retrieved from public databases revealed an extensive variation in genome sizes (6.4- 358.7 kb) and guanine plus cytosine (GC) content (35.5-65.4%) and a linear correlation between the genome size and the number of open reading frames (ORFs). We used three approaches to compare the phage genomes. The NUCmer/MUMmer genome alignment tool was used to evaluate linkages and correlations based on nucleotide identity between genomes. Multiple sequence alignment was performed to calculate genome average nucleotide identity using the Kalgin program. Finally, genome synteny was explored using dot plot analysis. We found that 90 phage genome sequences grouped into 17 distinct clusters while the remaining 52 genomes showed no close relationships with the other phage genomes and are identified as singletons. We generated genome maps using nucleotide and amino acid sequences which allowed protein-coding genes to be sorted into phamilies (phams) using the Phamerator software. Out of 5796 total assigned phamilies, one phamily was observed to be dominant and was found in 49 prophages, or 34.5% of the 142 phages in our collection. A majority of the phamilies, 4330 out of 5796 (74.7%), occurred in just one prophage underscoring the high degree of diversity among Salmonella bacteriophages. Conclusions: Based on nucleotide and amino acid sequences, a high diversity was found among Salmonella bacteriophages which validate the use of prophage sequence analysis as a highly discriminatory subtyping tool for Salmonella. Thorough understanding of the conservation and variation of prophage genomic characteristics will facilitate their rational design and use as tools for bacterial strain construction, vector development and as anti-bacterial agents.

This study identifies a strain of Salmonella enterica subspecies enterica serovar Enteritidis that harbors a highly unusual virulence plasmid. During the characterisation of a group of S. Enteritidis isolates, 10 isolates recovered from Canadian duck production facilities, of which seven were phage type 9b and three were closely related atypical phage types, failed detection by a PCR targeting the prot6e gene, a marker located on the virulence plasmid often employed for identification of this serovar. Comparison to prot6e+ isolates by several standard genetic typing tools, further revealed their distinctive genomic makeup. Both short read and long read whole genome sequencing were completed on six of these isolates. In addition to loss of the prot6e gene, the virulence plasmid of each isolate was found to be exceptionally large (86.5 Kb) due to a 28 Kb insertion of S. Typhimurium plasmid sequence that encodes multiple genes of the incF operon. Interrogation of the chromosome sequence data of these isolates using a SNP-based typing tool and MLST both indicated their close genetic relatedness. One additional isolate carrying this plasmid was identified in an in-house collection of S. Enteritidis isolates. Finally, the identification of this unusual plasmid sequence in additional isolates submitted to public repositories of Salmonella sequence data was explored. All these analyses indicated that a very distinctive but rarely reported strain of S. Enteritidis was widely distributed across North America and the United Kingdom with one additional report involving a case from Brazil. With increased use of genetic methods for Salmonella identification, the loss of the prot6e sequence may confound correct identification of this serovar while also potentially altering the mode of transmission to humans given the gene’s role in facilitating propagation of this bacterium in eggs. Accordingly, this strain may present certain challenges with respect to public health investigations. Our studies also suggest this strain is often associated with duck hosts thereby providing a possible mechanism by which this strain has spread over an extensive geographical area.

Complete genome sequences of eight isolates of Salmonella enterica subsp. enterica from Canadian wild birds were determined by MinION and Illumina MiSeq sequencing. Assembled chromosomes had an average size of 4,833,662 bp. Salmonella enterica serovar Worthington obtained from partridge and quail carried 267-kb plasmids, which contained multiple antimicrobial resistance genes.

The masked birch caterpillar, Drepana arcuata Walker (Lepidoptera: Drepanidae), and other Drepanoidea (Lepidoptera) species are excellent organisms for investigating the function and evolution of vibratory communication and sociality in caterpillars. We present a de novo assembled draft genome and functional annotation for D. arcuata, using a combination of short and long sequencing reads generated by Illumina HiSeq X and Oxford Nanopore Technologies (ONT) MinION sequencing platforms, respectively. A total of 460,694,612 150bp paired-end Illumina and 395,890 ONT raw reads were assembled into 11,493 scaffolds spanning a genome size of 270.5Mb. The resulting D. arcuata genome has a GC content of 38.79%, repeat content of 8.26%, is 86.5% complete based on Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment, and comprises 10,398 predicted protein-coding genes. These data represent the first genomic resources for the lepidopteran superfamily Drepanoidea. Although the order Lepidoptera comprises numerous ecologically and economically important species, assembled genomes and annotations are available for < 1% of the total species. These data can be further utilized for research on Lepidoptera genomics as well as on the function and evolution of vibratory communication and sociality in larval insects.

The complete genome sequences of 12 isolates of the rare Salmonella enterica serovar Adjame were determined by combining Nanopore and Illumina sequence reads. Chromosome sizes ranged from 4,597,011 bp to 4,678,052 bp, and the GC content was 52.3%. A virulent plasmid of 87,433 bp was found in only one isolate.

Non-typhoidal Salmonellae (NTS) belong to Salmonella enterica subspecies enterica and are common causes of foodborne illnesses in humans.Diarrhea is a common symptom but infection occasionally results in life-threatening systemic involvement.One member of the group, S. enterica subspecies enterica serovar Typhimurium has been extensively studied in live animal models particularly mice and cattle, leading to a better understanding of the pathogenesis of NTS and the development of diarrhea, respectively.This comprehensive review provides an insight into the genetic regulation of over 200 virulence determinants and their involvement in the four steps of Salmonella pathogenesis, namely: attachment, invasion, macrophage survival and replication, and systemic dissemination.There is, however, a paucity of information on the functions of some virulence factors present on the Salmonella pathogenicity islands (SPIs).The emergence of next generation sequencing (NGS) technology and the availability of more bacterial genomes should provide further insights into the biology of virulence determinants, mechanisms of NTS pathogenesis and host adaptation of Salmonella.The new knowledge should translate into improvement and innovations in food safety, and control of salmonellosis as well as better understanding of zoonotic infections in the context of One Health capturing the risks to humans, animals and the environment.

The rapid detection of foodborne microbial pathogens contaminating fresh fruits and vegetables during the intervening period between harvest and consumption could revolutionize microbial quality assurance of food usually consumed raw and those with a limited shelf life. We have developed a sensitive, shotgun whole genome sequencing protocol capable of detecting as few as 1 colony forming unit (cfu) of Salmonella enterica serovar Typhimurium spiked on 25 g of lettuce. The Ion Torrent sequencing platform was used to generate reads of globally amplified DNA from microbes recovered from the surface of lettuce followed by bioinformatic analyses of the nucleotide sequences to detect the presence of Salmonella. The test is rapid and sensitive, and appropriate for testing perishable foods, and those consumed raw, for Salmonella contamination. The test has the potential to be universally applicable to any microbial contaminant on lettuce as long as a suitable bioinformatics pipeline is available and validated. A universal test is expected to pave the way for preventive and precision food safety and the re-shaping of the entire spectrum of food safety investigations from the current disease-limiting, reactive procedure to a proactive, disease prevention process.