Assessment of plasmids for relating the 2020 Salmonella enterica serovar Newport onion outbreak to farms implicated by the outbreak investigation.

BACKGROUND: The Salmonella enterica serovar Newport red onion outbreak of 2020 was the largest foodborne outbreak of Salmonella in over a decade. The epidemiological investigation suggested two farms as the likely source of contamination. However, single nucleotide polymorphism (SNP) analysis of the whole genome sequencing data showed that none of the Salmonella isolates collected from the farm regions were linked to the clinical isolates-preventing the use of phylogenetics in source identification. Here, we explored an alternative method for analyzing the whole genome sequencing data driven by the hypothesis that if the outbreak strain had come from the farm regions, then the clinical isolates would disproportionately contain plasmids found in isolates from the farm regions due to horizontal transfer. RESULTS: SNP analysis confirmed that the clinical isolates formed a single, nearly-clonal clade with evidence for ancestry in California going back a decade. The clinical clade had a large core genome (4,399 genes) and a large and sparsely distributed accessory genome (2,577 genes, at least 64% on plasmids). At least 20 plasmid types occurred in the clinical clade, more than were found in the literature for Salmonella Newport. A small number of plasmids, 14 from 13 clinical isolates and 17 from 8 farm isolates, were found to be highly similar (> 95% identical)-indicating they might be related by horizontal transfer. Phylogenetic analysis was unable to determine the geographic origin, isolation source, or time of transfer of the plasmids, likely due to their promiscuous and transient nature. However, our resampling analysis suggested that observing a similar number and combination of highly similar plasmids in random samples of environmental Salmonella enterica within the NCBI Pathogen Detection database was unlikely, supporting a connection between the outbreak strain and the farms implicated by the epidemiological investigation. CONCLUSION: Horizontally transferred plasmids provided evidence for a connection between clinical isolates and the farms implicated as the source of the outbreak. Our case study suggests that such analyses might add a new dimension to source tracking investigations, but highlights the need for detailed and accurate metadata, more extensive environmental sampling, and a better understanding of plasmid molecular evolution.

Genomic analysis of Listeria monocytogenes from US food processing environments reveals a high prevalence of QAC efflux genes but limited evidence of their contribution to environmental persistence.

BACKGROUND: Quaternary ammonium compound (QAC) efflux genes increase the minimum inhibitory concentration of Listeria monocytogenes (Lm) to benzalkonium chloride sanitizer, but the contribution of these genes to persistence in food processing environments is unclear. The goal of this study was to leverage genomic data and associated metadata for 4969 Lm isolates collected between 1999 and 2019 to: (1) evaluate the prevalence of QAC efflux genes among Lm isolates from diverse US food processors, (2) use comparative genomic analyses to assess confounding factors, such as clonal complex identity and stress tolerance genotypes, and (3) identify patterns in QAC efflux gene gain and loss among persistent clones within specific facilities over time. RESULTS: The QAC efflux gene cassette bcrABC was present in nearly half (46%) of all isolates. QAC efflux gene prevalence among isolates was associated with clonal complex (𝛘2 < 0.001) and clonal complex was associated with the facility type (𝛘2 < 0.001). Consequently, changes in the prevalence of QAC efflux genes within individual facilities were generally attributable to changes in the prevalence of specific clonal complexes. Additionally, a GWAS and targeted BLAST search revealed that clonal complexes with a high prevalence of QAC efflux genes commonly possessed other stress tolerance genes. For example, a high prevalence of bcrABC in a clonal complex was significantly associated with the presence of the SSI-1 gene cluster (p < 0.05). QAC efflux gene gain and loss were both observed among persistent populations of Lm in individual facilities, suggesting a limited direct role for QAC efflux genes as predictors of persistence. CONCLUSION: This study suggests that although there is evidence that QAC efflux genes are part of a suite of adaptations common among Lm isolated from some food production environments, these genes may be neither sufficient nor necessary to enhance persistence. This is a crucial distinction for decision making in the food industry. For example, changes to sanitizer regimen targeting QAC tolerance would not address other contributing genetic or non-genetic factors, such as equipment hygienic design which physically mediates sanitizer exposure.

Interpretative Labor and the Bane of Nonstandardized Metadata in Public Health Surveillance and Food Safety.

Open-source DNA sequence databases have long been touted as beneficial to public health, including the facilitation of earlier detection and response to infectious disease outbreaks. Of critical importance to harnessing these benefits is the metadata that describe general and other domain-specific attributes (eg, collection location, isolate type) of a sample. Unlike the sequence data, metadata are often incomplete and lack adherence to an international standard. Here, we describe the problem posed by such variable and incomplete metadata in terms of interpretative labor costs (the time and energy necessary to make sense of the signal in the genetic data) and the impact such metadata have on foodborne outbreak detection and response. Improving the quality of sequence-associated metadata would allow for earlier detection of emerging food safety hazards and allow faster response to foodborne outbreaks.

Phylogenetic and Biogeographic Patterns of Vibrio parahaemolyticus Strains from North America Inferred from Whole-Genome Sequence Data.

Vibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States. The draft genomes of 132 North American clinical and oyster V. parahaemolyticus isolates were sequenced to investigate their phylogenetic and biogeographic relationships. The majority of oyster isolate sequence types (STs) were from a single harvest location; however, four were identified from multiple locations. There was population structure along the Gulf and Atlantic Coasts of North America, with what seemed to be a hub of genetic variability along the Gulf Coast, with some of the same STs occurring along the Atlantic Coast and one shared between the coastal waters of the Gulf and those of Washington State. Phylogenetic analyses found nine well-supported clades. Two clades were composed of isolates from both clinical and oyster sources. Four were composed of isolates entirely from clinical sources, and three were entirely from oyster sources. Each single-source clade consisted of one ST. Some human isolates lack tdh, trh, and some type III secretion system (T3SS) genes, which are established virulence genes of V. parahaemolyticus Thus, these genes are not essential for pathogenicity. However, isolates in the monophyletic groups from clinical sources were enriched in several categories of genes compared to those from monophyletic groups of oyster isolates. These functional categories include cell signaling, transport, and metabolism. The identification of genes in these functional categories provides a basis for future in-depth pathogenicity investigations of V. parahaemolyticusIMPORTANCEVibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States and is frequently associated with shellfish consumption. This study contributes to our knowledge of the biogeography and functional genomics of this species around North America. STs shared between the Gulf Coast and the Atlantic seaboard as well as Pacific waters suggest possible transport via oceanic currents or large shipping vessels. STs frequently isolated from humans but rarely, if ever, isolated from the environment are likely more competitive in the human gut than other STs. This could be due to additional functional capabilities in areas such as cell signaling, transport, and metabolism, which may give these isolates an advantage in novel nutrient-replete environments such as the human gut.

Investigating the Distribution of Strains of Erwinia amylovora and Streptomycin Resistance in Apple Orchards in New York Using Clustered Regularly Interspaced Short Palindromic Repeat Profiles: A 6-Year Follow-Up.

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY. Motivated by new grower reports of control failures, we conducted a follow-up investigation of the distribution of SmR and E. amylovora strains for major apple production regions of NY over the last 6 years (2015 to 2020). Characterization of clustered regularly interspaced short palindromic repeat (CRISPR) profiles revealed that a few "cosmopolitan" strains were widely prevalent across regions, whereas many other "resident" strains were confined to one location. In addition, we uncovered novel CRISPR profile diversity in all investigated regions. SmR E. amylovora was detected only in a small area spanning two counties from 2017 to 2020 and was always associated with one CRISPR profile (41:23:38), which matched the profile of SmR E. amylovora, discovered in 2002. This suggests the original SmR E. amylovora was never fully eradicated and went undetected because of several seasons of low disease pressure in this region. Investigation of several representative isolates under controlled greenhouse conditions indicated significant differences in aggressiveness on 'Gala' apples. Potential implications of strain differences include the propensity of strains to become distributed across wide geographic regions and associated resistance management practices. Results from this work will directly influence sustainable fire blight management recommendations for commercial apple industries in NY state and other regions.

Outbreak of Tattoo-associated Nontuberculous Mycobacterial Skin Infections.

BACKGROUND: On 29 April 2015, the Florida Department of Health in Miami-Dade County (DOH Miami-Dade) was notified by a local dermatologist of 3 patients with suspected nontuberculous mycobacterial (NTM) infection after receiving tattoos at a local tattoo studio. METHODS: DOH Miami-Dade conducted interviews and offered testing, described below, to tattoo studio clients reporting rashes. Culture of clinical isolates and identification were performed at the Florida Bureau of Public Health Laboratories. Characterization of NTM was performed by the Centers for Disease Control and Prevention and the US Food and Drug Administration (FDA), respectively. Whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analyses were used to construct a phylogeny among 21 Mycobacterium isolates at the FDA. RESULTS: Thirty-eight of 226 interviewed clients were identified as outbreak-associated cases. Multivariate logistic regression revealed that individuals who reported gray tattoo ink in their tattoos were 8.2 times as likely to report a rash (95% confidence interval, 3.1-22.1). Multiple NTM species were identified in clinical and environmental specimens. Phylogenetic results from environmental samples and skin biopsies indicated that 2 Mycobacterium fortuitum isolates (graywash ink and a skin biopsy) and 11 Mycobacterium abscessus isolates (5 from the implicated bottle of graywash tattoo ink, 2 from tap water, and 4 from skin biopsies) were indistinguishable. In addition, Mycobacterium chelonae was isolated from 5 unopened bottles of graywash ink provided by 2 other tattoo studios in Miami-Dade County. CONCLUSIONS: WGS and SNP analyses identified the tap water and the bottle of graywash tattoo ink as the sources of the NTM infections.

Mating system divergence affects the distribution of sequence diversity within and among populations of recently diverged subspecies of Clarkia xantiana (Onagraceae).

PREMISE OF THE STUDY: The population biology of outcrossing and self-fertilizing taxa is thought to differ because of the advantage that selfers have in colonizing unoccupied sites where mates and pollinators may be limiting (Baker's Law). This reduced tendency for outcrossers to colonize new sites, along with their greater dependence on pollinators to disperse pollen, has the potential to differently influence the genetic diversity and structure of outcrossing and selfing populations. METHODS: We conducted a comparative population genetic study of two sister outcrossing and selfing subspecies of Clarkia xantiana that have very recently diverged. We used DNA sequence variation (>40 kb from eight nuclear loci) from large samples of individuals from 14 populations to assess geographic patterns of genetic diversity and make inferences about the demographic and colonization histories of each subspecies. KEY RESULTS: We show that sequence variation is strongly reduced across all selfing populations. The demographic history of selfing populations exhibits recent colonization bottlenecks, whereas such bottlenecks are rarely observed for the outcrosser. The greater effect of genetic drift in the selfer has resulted in strong population genetic structure, but with no pattern of isolation by distance. By contrast, the stronger effect of gene flow in the outcrosser has resulted in considerably less structure, but a significant pattern of isolation by distance. CONCLUSIONS: Taken together, our results suggest that selfing populations are not at migration-drift equilibrium, are affected by strong episodes of genetic drift during colonization, and experience little or no subsequent gene flow from other populations after those founder events.

Characterization and evolution of Salmonella CRISPR-Cas systems.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12 % of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9 %) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

Characterization and evolution of Salmonella CRISPR-Cas systems.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing.

BACKGROUND: Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq. RESULTS: Time zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples. CONCLUSIONS: Our cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve detection sensitivity for foodborne enteric pathogens.

The impact of systemic and copper pesticide applications on the phyllosphere microflora of tomatoes.

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

Prospective modeling and estimating the epidemiologically informative match rate within large foodborne pathogen genomic databases.

OBJECTIVES: Much has been written about the utility of genomic databases to public health. Within food safety these databases contain data from two types of isolates-those from patients (i.e., clinical) and those from non-clinical sources (e.g., a food manufacturing environment). A genetic match between isolates from these sources represents a signal of interest. We investigate the match rate within three large genomic databases (Listeria monocytogenes, Escherichia coli, and Salmonella) and the smaller Cronobacter database; the databases are part of the Pathogen Detection project at NCBI (National Center for Biotechnology Information). RESULTS: Currently, the match rate of clinical isolates to non-clinical isolates is 33% for L. monocytogenes, 46% for Salmonella, and 7% for E. coli. These match rates are associated with several database features including the diversity of the organism, the database size, and the proportion of non-clinical BioSamples. Modeling match rate via logistic regression showed relatively good performance. Our prediction model illustrates the importance of populating databases with non-clinical isolates to better identify a match for clinical samples. Such information should help public health officials prioritize surveillance strategies and show the critical need to populate fledgling databases (e.g., Cronobacter sakazakii).

Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populus and the plant kingdom.

BACKGROUND: Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. RESULTS: We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. CONCLUSION: In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may function in nutrient sensing and/or signaling. These proteins are members of Group I NP-like proteins, which are widely distributed in many plant taxa. We conclude that NP-like proteins may function in plants, although this function is undefined.

Baseline survey of the anatomical microbial ecology of an important food plant: Solanum lycopersicum (tomato).

BACKGROUND: Research to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop. RESULTS: DNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe "native" bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes. CONCLUSIONS: Distinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Dominant fungal taxa that could be identified to genus with 18S amplicons included Hypocrea, Aureobasidium and Cryptococcus. No definitive presence of Salmonella could be confirmed in any of the plant samples, although 16S sequences suggested that closely related genera were present on leaves, fruits and roots.

Performance of methods for SARS-CoV-2 variant detection and abundance estimation within mixed population samples.

Background: The accurate identification of SARS-CoV-2 (SC2) variants and estimation of their abundance in mixed population samples (e.g., air or wastewater) is imperative for successful surveillance of community level trends. Assessing the performance of SC2 variant composition estimators (VCEs) should improve our confidence in public health decision making. Here, we introduce a linear regression based VCE and compare its performance to four other VCEs: two re-purposed DNA sequence read classifiers (Kallisto and Kraken2), a maximum-likelihood based method (Lineage deComposition for Sars-Cov-2 pooled samples (LCS)), and a regression based method (Freyja). Methods: We simulated DNA sequence datasets of known variant composition from both Illumina and Oxford Nanopore Technologies (ONT) platforms and assessed the performance of each VCE. We also evaluated VCEs performance using publicly available empirical wastewater samples collected for SC2 surveillance efforts. Bioinformatic analyses were performed with a custom NextFlow workflow (C-WAP, CFSAN Wastewater Analysis Pipeline). Relative root mean squared error (RRMSE) was used as a measure of performance with respect to the known abundance and concordance correlation coefficient (CCC) was used to measure agreement between pairs of estimators. Results: Based on our results from simulated data, Kallisto was the most accurate estimator as it had the lowest RRMSE, followed by Freyja. Kallisto and Freyja had the most similar predictions, reflected by the highest CCC metrics. We also found that accuracy was platform and amplicon panel dependent. For example, the accuracy of Freyja was significantly higher with Illumina data compared to ONT data; performance of Kallisto was best with ARTICv4. However, when analyzing empirical data there was poor agreement among methods and variations in the number of variants detected (e.g., Freyja ARTICv4 had a mean of 2.2 variants while Kallisto ARTICv4 had a mean of 10.1 variants). Conclusion: This work provides an understanding of the differences in performance of a number of VCEs and how accurate they are in capturing the relative abundance of SC2 variants within a mixed sample (e.g., wastewater). Such information should help officials gauge the confidence they can have in such data for informing public health decisions.

Application of quasimetagenomics methods to define microbial diversity and subtype Listeria monocytogenes in dairy and seafood production facilities.

IMPORTANCE: In developed countries, the human diet is predominated by food commodities, which have been manufactured, processed, and stored in a food production facility. Little is known about the application of metagenomic sequencing approaches for detecting foodborne pathogens, such as L. monocytogenes, and characterizing microbial diversity in food production ecosystems. In this work, we investigated the utility of 16S rRNA amplicon and quasimetagenomic sequencing for the taxonomic and phylogenetic classification of Listeria culture enrichments of environmental swabs collected from dairy and seafood production facilities. We demonstrated that single-nucleotide polymorphism (SNP) analyses of L. monocytogenes metagenome-assembled genomes (MAGs) from quasimetagenomic data sets can achieve similar resolution as culture isolate whole-genome sequencing. To further understand the impact of genome coverage on MAG SNP cluster resolution, an in silico downsampling approach was employed to reduce the percentage of target pathogen sequence reads, providing an initial estimate of required MAG coverage for subtyping resolution of L. monocytogenes.

A longitudinal study to examine the influence of farming practices and environmental factors on pathogen prevalence using structural equation modeling.

The contamination of fresh produce with foodborne pathogens has been an on-going concern with outbreaks linked to these commodities. Evaluation of farm practices, such as use of manure, irrigation water source, and other factors that could influence pathogen prevalence in the farming environment could lead to improved mitigation strategies to reduce the potential for contamination events. Soil, water, manure, and compost were sampled from farms in Ohio and Georgia to identify the prevalence of Salmonella, Listeria monocytogenes (Lm), Campylobacter, and Shiga-toxin-producing Escherichia coli (STEC), as well as Arcobacter, an emerging human pathogen. This study investigated agricultural practices to determine which influenced pathogen prevalence, i.e., the percent positive samples. These efforts identified a low prevalence of Salmonella, STEC, and Campylobacter in soil and water (< 10%), preventing statistical modeling of these pathogens. However, Lm and Arcobacter were found in soil (13 and 7%, respectively), manure (49 and 32%, respectively), and water samples (18 and 39%, respectively) at a comparatively higher prevalence, suggesting different dynamics are involved in their survival in the farm environment. Lm and Arcobacter prevalence data, soil chemical characteristics, as well as farm practices and weather, were analyzed using structural equation modeling to identify which factors play a role, directly or indirectly, on the prevalence of these pathogens. These analyses identified an association between pathogen prevalence and weather, as well as biological soil amendments of animal origin. Increasing air temperature increased Arcobacter and decreased Lm. Lm prevalence was found to be inversely correlated with the use of surface water for irrigation, despite a high Lm prevalence in surface water suggesting other factors may play a role. Furthermore, Lm prevalence increased when the microbiome's Simpson's Diversity Index decreased, which occurred as soil fertility increased, leading to an indirect positive effect for soil fertility on Lm prevalence. These results suggest that pathogen, environment, and farm management practices, in addition to produce commodities, all need to be considered when developing mitigation strategies. The prevalence of Arcobacter and Lm versus the other pathogens suggests that multiple mitigation strategies may need to be employed to control these pathogens.

Multinational Outbreak of Listeria monocytogenes Infections Linked to Enoki Mushrooms Imported from the Republic of Korea 2016-2020.

Keeping the global food supply safe necessitates international collaborations between countries. Health and regulatory agencies routinely communicate during foodborne illness outbreaks, allowing partners to share investigational evidence. A 2016-2020 outbreak of Listeria monocytogenes infections linked to imported enoki mushrooms required a multinational collaborative investigation among the United States, Canada, Australia, and France. Ultimately, this outbreak included 48 ill people, 36 in the United States and 12 in Canada, and was linked to enoki mushrooms sourced from one manufacturer located in the Republic of Korea. Epidemiologic, laboratory, and traceback evidence led to multiple regulatory actions, including extensive voluntary recalls by three firms in the United States and one firm in Canada. In the United States and Canada, the Korean manufacturer was placed on import alert while other international partners provided information about their respective investigations and advised the public not to eat the recalled enoki mushrooms. The breadth of the geographic distribution of this outbreak emphasizes the global reach of the food industry. This investigation provides a powerful example of the impact of national and international coordination of efforts to respond to foodborne illness outbreaks and protect consumers. It also demonstrates the importance of fast international data sharing and collaboration in identifying and stopping foodborne outbreaks in the global community. Additionally, it is a meaningful example of the importance of food sampling, testing, and integration of sequencing results into surveillance databases.

Phylogeography of speciation: allopatric divergence and secondary contact between outcrossing and selfing Clarkia.

The origins of hybrid zones between parapatric taxa have been of particular interest for understanding the evolution of reproductive isolation and the geographic context of species divergence. One challenge has been to distinguish between allopatric divergence (followed by secondary contact) versus primary intergradation (parapatric speciation) as alternative divergence histories. Here, we use complementary phylogeographic and population genetic analyses to investigate the recent divergence of two subspecies of Clarkia xantiana and the formation of a hybrid zone within the narrow region of sympatry. We tested alternative phylogeographic models of divergence using approximate Bayesian computation (ABC) and found strong support for a secondary contact model and little support for a model allowing for gene flow throughout the divergence process (i.e. primary intergradation). Two independent methods for inferring the ancestral geography of each subspecies, one based on probabilistic character state reconstructions and the other on palaeo-distribution modelling, also support a model of divergence in allopatry and range expansion leading to secondary contact. The membership of individuals to genetic clusters suggests geographic substructure within each taxon where allopatric and sympatric samples are primarily found in separate clusters. We also observed coincidence and concordance of genetic clines across three types of molecular markers, which suggests that there is a strong barrier to gene flow. Taken together, our results provide evidence for allopatric divergence followed by range expansion leading to secondary contact. The location of refugial populations and the directionality of range expansion are consistent with expectations based on climate change since the last glacial maximum. Our approach also illustrates the utility of combining phylogeographic hypothesis testing with species distribution modelling and fine-scale population genetic analyses for inferring the geography of the divergence process.