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.

Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

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.

Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce.

Lettuce is associated with seasonal outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections. Little is known about how various biotic and abiotic factors affect the lettuce microbiome, which in turn impacts STEC colonization. We characterized the lettuce phyllosphere and surface soil bacterial, fungal, and oomycete communities at harvest in late-spring and -fall in California using metagenomics. Harvest season and field type, but not cultivar, significantly influenced the microbiome composition of leaves and surface soil near plants. Phyllosphere and soil microbiome compositions were correlated with specific weather factors. The relative abundance of Enterobacteriaceae, but not E. coli, was enriched on leaves (5.2%) compared to soil (0.4%) and correlated positively with minimum air temperature and wind speed. Co-occurrence networks revealed seasonal trends in fungi-bacteria interactions on leaves. These associations represented 39%-44% of the correlations between species. All significant E. coli co-occurrences with fungi were positive, while all negative associations were with bacteria. A large proportion of the leaf bacterial species was shared with those in soil, indicating microbiome transmission from the soil surface to the canopy. Our findings provide new insight into factors that shape lettuce microbial communities and the microbial context of foodborne pathogen immigration events in the lettuce phyllosphere.

Phylogenomic Analysis of Salmonella enterica subsp. enterica Serovar Bovismorbificans from Clinical and Food Samples Using Whole Genome Wide Core Genes and kmer Binning Methods to Identify Two Distinct Polyphyletic Genome Pathotypes

Salmonella enterica subsp. enterica serovar Bovismorbificans has caused multiple outbreaks involving the consumption of produce, hummus, and processed meat products worldwide. To elucidate the intra-serovar genomic structure of S. Bovismorbificans, a core-genome analysis with 2690 loci (based on 150 complete genomes representing Salmonella enterica serovars developed as part of this study) and a k-mer-binning based strategy were carried out on 95 whole genome sequencing (WGS) assemblies from Swiss, Canadian, and USA collections of S. Bovismorbificans strains from foodborne infections. Data mining of a digital DNA tiling array of legacy SARA and SARB strains was conducted to identify near-neighbors of S. Bovismorbificans. The core genome analysis and the k-mer-binning methods identified two polyphyletic clusters, each with emerging evolutionary properties. Four STs (2640, 142, 1499, and 377), which constituted the majority of the publicly available WGS datasets from >260 strains analyzed by k-mer-binning based strategy, contained a conserved core genome backbone with a different evolutionary lineage as compared to strains comprising the other cluster (ST150). In addition, the assortment of genotypic features contributing to pathogenesis and persistence, such as antimicrobial resistance, prophage, plasmid, and virulence factor genes, were assessed to understand the emerging characteristics of this serovar that are relevant clinically and for food safety concerns. The phylogenomic profiling of polyphyletic S. Bovismorbificans in this study corresponds to intra-serovar variations observed in S. Napoli and S. Newport serovars using similar high-resolution genomic profiling approaches and contributes to the understanding of the evolution and sequence divergence of foodborne Salmonellae. These intra-serovar differences may have to be thoroughly understood for the accurate classification of foodborne Salmonella strains needed for the uniform development of future food safety mitigation strategies.

Draft Genome Sequences of 18 Streptococcus Strains Isolated from Live Dietary Supplements and Cultured Food Products.

We present the genome sequences of 18 Streptococcus isolates from 8 different dietary supplements and 9 cultured food products. Strains from this species naturally colonize the human mouth and upper respiratory tract. Studies have shown that S. thermophilus and S. salivarius strains confer oral health benefits to their host with little to no risk of pathogenic infection.

Draft Genome Sequences of Nine Bacillus Strains and Two Weizmannia Strains Isolated from Live Dietary Supplements and a Cultured Food Product.

We present the genome sequences of nine Bacillus isolates and two Weizmannia isolates from 10 different dietary supplements and one cultured food product. Strains of these species have been associated with health benefits when ingested by humans, due to their ability to survive the stomach's acidic environment and colonize the intestinal tract.

Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California.

BACKGROUND: Lettuce is linked to recurrent outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections, the seasonality of which remains unresolved. Infections have occurred largely from processed lettuce, which undergoes substantial physiological changes during storage. We investigated the microbiome and STEC O157:H7 (EcO157) colonization of fresh-cut lettuce of two cultivars with long and short shelf life harvested in the spring and fall in California and stored in modified atmosphere packaging (MAP) at cold and warm temperatures. RESULTS: Inoculated EcO157 declined significantly less on the cold-stored cultivar with short shelf life, while multiplying rapidly at 24 °C independently of cultivar. Metagenomic sequencing of the lettuce microbiome revealed that the pre-storage bacterial community was variable but dominated by species in the Erwiniaceae and Pseudomonadaceae. After cold storage, the microbiome composition differed between cultivars, with a greater relative abundance (RA) of Erwiniaceae and Yersiniaceae on the cultivar with short shelf life. Storage at 24 °C shifted the microbiome to higher RAs of Erwiniaceae and Enterobacteriaceae and lower RA of Pseudomonadaceae compared with 6 °C. Fall harvest followed by lettuce deterioration were identified by recursive partitioning as important factors associated with high EcO157 survival at 6 °C, whereas elevated package CO2 levels correlated with high EcO157 multiplication at 24 °C. EcO157 population change correlated with the lettuce microbiome during 6 °C storage, with fall microbiomes supporting the greatest EcO157 survival on both cultivars. Fall and spring microbiomes differed before and during storage at both temperatures. High representation of Pantoea agglomerans was a predictor of fall microbiomes, lettuce deterioration, and enhanced EcO157 survival at 6 °C. In contrast, higher RAs of Erwinia persicina, Rahnella aquatilis, and Serratia liquefaciens were biomarkers of spring microbiomes and lower EcO157 survival. CONCLUSIONS: The microbiome of processed MAP lettuce evolves extensively during storage. Under temperature abuse, high CO2 promotes a lettuce microbiome enriched in taxa with anaerobic capability and EcO157 multiplication. In cold storage, our results strongly support a role for season and lettuce deterioration in EcO157 survival and microbiome composition, suggesting that the physiology and microbiomes of fall- and spring-harvested lettuce may contribute to the seasonality of STEC outbreaks associated with lettuce grown in coastal California.

Development and validation of an improved method for the detection of Salmonella in cinnamon bark and oregano leaves using the adsorbent beta zeolite in the pre-enrichment media.

The detection of Salmonella in spices is challenging due to the presence of antibacterial components. In this study, we evaluated the use of an adsorbent beta zeolite in pre-enrichment media to improve the recovery of Salmonella from cinnamon bark and oregano leaves. Samples (25 g) were spiked with varying levels of S. Montevideo or S. Senftenberg. After 2 weeks of stabilization at RT, betazeolite was added to cinnamon and oregano samples prior to the addition of 225 mL or 475 mL of pre-enrichment media, respectively. Detection sensitivity and rate of the test method were compared to the FDA Bacteriological Analytical Manual (BAM) method which requires the use of 2.5 L pre-enrichment broth. While Salmonella could not be detected in the test method using the reduced volume of pre-enrichment media alone, the addition of beta zeolite resulted in a positivity rate of 62% and 72.6% for cinnamon bark and oregano leaves respectively (all spike levels and both serovars combined). Furthermore, while there were differences in the LOD50 compared to the BAM method, there was no significant difference in the minimum level of detection between the betazeolite and the BAM methods. Our results demonstrate that the use of betazeolite in the pre-enrichment media offers a method with reduced media volumes without compromising on the sensitivity or efficiency of Salmonella detection in cinnamon bark and oregano leaves.

Comparative Transcriptomics of Shiga Toxin-Producing and Commensal Escherichia coli and Cytokine Responses in Colonic Epithelial Cell Culture Infections.

Ingestion of Shiga toxin-producing Escherichia coli (STEC) can result in a range of illness severity from asymptomatic to hemorrhagic colitis and death; thus risk assessment of STEC strains for human pathogenicity is important in the area of food safety. Illness severity depends in part on the combination of virulence genes carried in the genome, which can vary between strains even of identical serotype. To better understand how core genes are regulated differently among strains and to identify possible novel STEC virulence gene candidates that could be added to the risk assessment repertoire, we used comparative transcriptomics to investigate global gene expression differences between two STEC strains associated with severe illness and a commensal E. coli strain during in vitro intestinal epithelial cell (IEC) infections. Additionally, we compared a wide array of concomitant cytokine levels produced by the IECs. The cytokine expression levels were examined for a pattern representing STEC pathogenicity; however, while one STEC strain appeared to elicit a proinflammatory response, infection by the other strain produced a pattern comparable to the commensal E. coli. This result may be explained by the significant differences in gene content and expression observed between the STEC strains. RNA-Seq analysis revealed considerable disparity in expression of genes in the arginine and tryptophan biosynthesis/import pathways between the STEC strains and the commensal E. coli strain, highlighting the important role some amino acids play in STEC colonization and survival. Contrasting differential expression patterns were observed for genes involved in respiration among the three strains suggesting that metabolic diversity is a strategy utilized to compete with resident microflora for successful colonization. Similar temporal expression results for known and putative virulence genes were observed in the STEC strains, revealing strategies used for survival prior to and after initial adherence to IECs. Additionally, three genes encoding hypothetical proteins located in mobile genetic elements were, after interrogation of a large set of E. coli genomes, determined to likely represent novel STEC virulence factors.

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010-2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this "pattern of circulation" has continued over decades.

Draft Genome Sequences of 20 Aspergillus flavus Isolates from Corn Kernels and Cornfield Soils in Louisiana.

Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.

Draft Genome Sequences of Isolates of Diverse Host Origin from the E. coli Reference Center at Penn State University.

Escherichia coli strains present a vast genomic diversity. We report the draft genome sequences of 1,000 isolates from the E. coli Reference Center at Penn State University. These strains were originally isolated from multiple animal and environmental sources over the past 50 years.

A loop-mediated isothermal amplification (LAMP) assay for the consensus detection of human pathogenic Campylobacter species.

Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.

Draft Genome Sequences of Alternaria Strains Isolated from Grapes and Apples.

Here, we report the draft genome sequences of Alternaria alternata, isolated from seedless grapes, and Alternaria arborescens and Alternaria atra, isolated from Red Delicious apples, all from the Washington, DC, area.

Insular Microbiogeography: Three Pathogens as Exemplars.

Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.

Microbiota of the Hickey Run Tributary of the Anacostia River.

Water from the Hickey Run Tributary of the Anacostia River is being collected quarterly (beginning August 2018) and analyzed to create high-resolution baseline taxonomic profiles of microbiota associated with this important aquatic ecosystem, which has a long history of exposure to residential and commercial effluents from Washington, DC. These United States National Arboretum Microbial Observatory data are available under NCBI BioProject number PRJNA498951.

Draft Genome Sequences of 12 Isolates from 3 Fusarium Species Recovered from Moldy Peanuts.

In this report, we announce the sequences of six genomes of Fusarium proliferatum (isolates MOD1-FUNGI8, -12, -13, -14, -15, and -19), four genomes of Fusarium oxysporum (MOD1-FUNGI9, -10, -11, and -16), and two genomes of the Fusarium incarnatum-Fusarium equiseti species complex (MOD1-FUNGI17 and MOD1-FUNGI18) isolated from moldy peanuts from the Washington, DC, area.

Global transcriptomic response of Listeria monocytogenes during growth on cantaloupe slices.

Understanding a pathogen's response to food environments is imperative to develop effective control strategies as well as to elucidate the impact of foods on virulence potential. The purpose of this study was to assess transcriptional response of Listeria monocytogenes after growth in cantaloupe, as well as its impact on survival in synthetic gastric fluid (SGF). The transcriptional profiles of L. monocytogenes grown in cantaloupe or Brain Heart Infusion (BHI) under refrigeration were compared by a custom-designed microarray. A total of 286 and 175 genes were significantly up- and down-regulated, respectively, in L. monocytogenes grown in cantaloupe as compared to BHI (fold change ≥ 2.5 and adj. P < 0.05). The majority of upregulated genes belonged to functions related to amino acid and nucleotide metabolism, flagellar biosynthesis, and iron acquisition, while most downregulated genes belonged to carbohydrate metabolism. Notably, the branched chain amino acid (BCAA: leucine, isoleucine, valine) biosynthesis operon was shown to be highly upregulated as well as the purine and pyrimidine biosynthesis pathways. Transcript levels of several stress- and virulence-related genes were significantly altered, implying an impact of growth in cantaloupe on the virulence potential of L. monocytogenes. Enhanced survival of L. monocytogenes in SGF following growth in cantaloupe further demonstrated the impact of cantaloupe-associated growth on the pathogen's subsequent response to a host relevant stress.