Unveiling the genomic landscape of Salmonella enterica serotypes Typhimurium, Newport, and Infantis in Latin American surface waters: a comparative analysis.

Surface waters are considered ecological habitats where Salmonella enterica can persist and disseminate to fresh produce production systems. This study aimed to explore the genomic profiles of S. enterica serotypes Typhimurium, Newport, and Infantis from surface waters in Chile, Mexico, and Brazil collected between 2019 and 2022. We analyzed the whole genomes of 106 S. Typhimurium, 161 S. Newport, and 113 S. Infantis isolates. Our phylogenetic analysis exhibited distinct groupings of isolates by their respective countries except for a notable case involving a Chilean S. Newport isolate closely related to two Mexican isolates, showing 4 and 13 single nucleotide polymorphisms of difference, respectively. The patterns of the most frequently detected antimicrobial resistance genes varied across countries and serotypes. A strong correlation existed between integron carriage and genotypic multidrug resistance (MDR) across serotypes in Chile and Mexico (R > 0.90, P < 0.01), while integron(s) were not detected in any of the Brazilian isolates. By contrast, we did not identify any strong correlation between plasmid carriage and genotypic MDR across diverse countries and serotypes.IMPORTANCEUnveiling the genomic landscape of S. enterica in Latin American surface waters is pivotal for ensuring public health. This investigation sheds light on the intricate genomic diversity of S. enterica in surface waters across Chile, Mexico, and Brazil. Our research also addresses critical knowledge gaps, pioneering a comprehensive understanding of surface waters as a reservoir for multidrug-resistant S. enterica. By integrating our understanding of integron carriage as biomarkers into broader MDR control strategies, we can also work toward targeted interventions that mitigate the emergence and dissemination of MDR in S. enterica in surface waters. Given its potential implications for food safety, this study emphasizes the critical need for informed policies and collaborative initiatives to address the risks associated with S. enterica in surface waters.

Evaluation of universal preenrichment broth and comparison of rapid molecular methods for the detection of Salmonella from spent sprout irrigation water (SSIW).

Sprouts and spent sprout irrigation water (SSIW) present unique challenges for the development of a Salmonella detection method in food matrices. This study aimed to compare universal preenrichment broth (UPB) and lactose broth (LB) as preenrichment media for cultural and rapid screening methods and to compare their abilities to recover Salmonella in SSIW samples from different sprout varieties (i.e., alfalfa, broccoli, and mung bean sprouts). The associated co-enriched microbiota with different sprout varieties using different preenrichment media were also examined using a quasi-metagenomic approach. The performance of media and detection methods was compared using the relative level of detection (RLOD) value, as recommended by ISO 16140-2:2016. The level of detection (LOD) for Salmonella culture method with UPB was similar to that with LB in low aerobic plate count (APC) background samples (the relative LOD, i.e., RLOD, was nearly 1 after adjusting for the effects of SSIW variety and serovar), but significantly lower than that with LB in high APC background samples (RLOD = 0.32). The LOD for Salmonella with selected rapid methods was comparable to each other (RLOD from 0.97 to 1.50) and to the culture method (RLOD from 0.69 to 1.03), and no significant difference was detected between preenrichment broths in low APC background samples with RLOD values between 0.76 and 1.04. In samples with a high APC background, however, a drastic difference in LOD was observed between methods and between preenrichment broths for each method. The RLOD ranged from 0.03 to 0.32 when UPB was compared to LB as preenrichment broth. The composition and relative abundance (RA) of co-enriched microbiota was affected by multiple factors including food matrices, preenrichment media and Salmonella contamination. Altogether, this study validated UPB as a better preenrichment broth than LB for the detection of Salmonella enterica from SSIW. This study also suggested UPB may also be an optimal preenrichment medium for rapid screening methods when APC level is high. The observation of potential exclusion of Salmonella in preenrichment through the overgrowth of competitive microflora from the quasi-metagenomic study provided novel information that may be used to further optimize preenrichment formulations.

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.

Selecting an Optimal Faraday Cage To Minimize Noise in Electrochemical Experiments.

The ubiquitous Faraday cage, an experimental component particularly essential for nanoelectrochemical measurements, is responsible for neutralizing noise introduced by electromagnetic interference (EMI). Faraday cage designs abound in the literature, often exhibiting varying thicknesses, mesh sizes, and base materials. The fact that the Faraday cage composition most often goes unreported underscores the fact that many electrochemical researchers assume a 100% EMI reduction for any given design. In this work, this assumption is challenged from a theoretical and empirical perspective by highlighting the physical principles producing the Faraday effect. A brief history of the Faraday cage and a simplified theoretical approach introduce fundamental considerations regarding optimal design properties. In practice, time-domain noise profiles and corresponding Fourier transform frequency domain information for custom-built Faraday cages reveal that maximally conductive cages provide more optimal EMI exclusion.

ACEstat: A DIY Guide to Unlocking the Potential of Integrated Circuit Potentiostats for Open-Source Electrochemical Analysis.

Miniaturization of analytical instrumentation is paramount to enabling convenient in-field sensing. The recent thrust in potentiostat miniaturization for electrochemical sensing and general use has led to the development of commercial application specific integrated circuits (ASICs) that pack all the power of a benchtop instrument into one 5 mm × 5 mm chip. While the capabilities of these integrated circuits far exceed those of open-source potentiostats in the literature, the activation barrier for their implementation requires extensive electrical and software engineering expertise to overcome. In order to more rapidly bring the utility of ASIC potentiostats to researchers, we present a low size, weight, power, and cost (Low SWaP-C) Army Corps of Engineers potentiostat (ACEstat) based on the widely available ADuCM355 offered by Analog Devices. This potentiostat is a streamlined and fully programmable device that leverages industry-leading integrated hardware to perform electrochemical measurements such as cyclic voltammetry, pulse voltammetry, and electrochemical impedance spectroscopy. The ACEstat enables control over a wide range of test parameters and displays results through an intuitive, open-source graphical user interface available on mobile devices and computers. In this report, we present an approachable, do-it-yourself guide to unlocking the capabilities of this integrated circuit potentiostat by outlining the fabrication and programming details necessary to facilitate electroanalysis. Furthermore, we demonstrate the practicality of this device by detecting 2,4,6-trinitrotoluene (TNT) in water at sub-mg/L detection limits, highlighting its potential for in-field use.

Genomic evidence of environmental and resident Salmonella Senftenberg and Montevideo contamination in the pistachio supply-chain.

Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed an in-depth retrospective data analysis of Salmonella associated with pistachios as well as a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed utilizing short-read and long-read sequencing technologies. The sequence data were analyzed using two methods: the FDA's Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multilocus Sequence Typing (wgMLST). The year-long storage study evaluated the survival of five strains of Salmonella on pistachios stored at 25 °C at 35% and 54% relative humidity (RH). Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) the use of metagenomic analysis is a novel tool for determining the composition of serovar survival in a cocktail inoculated storage study.

The Persistence of Bacterial Pathogens in Surface Water and Its Impact on Global Food Safety.

Water is vital to agriculture. It is essential that the water used for the production of fresh produce commodities be safe. Microbial pathogens are able to survive for extended periods of time in water. It is critical to understand their biology and ecology in this ecosystem in order to develop better mitigation strategies for farmers who grow these food crops. In this review the prevalence, persistence and ecology of four major foodborne pathogens, Shiga toxin-producing Escherichia coli (STEC), Salmonella, Campylobacter and closely related Arcobacter, and Listeria monocytogenes, in water are discussed. These pathogens have been linked to fresh produce outbreaks, some with devastating consequences, where, in a few cases, the contamination event has been traced to water used for crop production or post-harvest activities. In addition, antimicrobial resistance, methods improvements, including the role of genomics in aiding in the understanding of these pathogens, are discussed. Finally, global initiatives to improve our knowledge base of these pathogens around the world are touched upon.

Whole genome sequencing and protein structure analyses of target genes for the detection of Salmonella.

Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T ↔ C (15 times) and A ↔ G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C → T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) → Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre2 and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and ß-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella.

Salmonella Genomics in Public Health and Food Safety.

The species Salmonella enterica comprises over 2,600 serovars, many of which are known to be intracellular pathogens of mammals, birds, and reptiles. It is now apparent that Salmonella is a highly adapted environmental microbe and can readily persist in a number of environmental niches, including water, soil, and various plant (including produce) species. Much of what is known about the evolution and diversity of nontyphoidal Salmonella serovars (NTS) in the environment is the result of the rise of the genomics era in enteric microbiology. There are over 340,000 Salmonella genomes available in public databases. This extraordinary breadth of genomic diversity now available for the species, coupled with widespread availability and affordability of whole-genome sequencing (WGS) instrumentation, has transformed the way in which we detect, differentiate, and characterize Salmonella enterica strains in a timely way. Not only have WGS data afforded a detailed and global examination of the molecular epidemiological movement of Salmonella from diverse environmental reservoirs into human and animal hosts, but they have also allowed considerable consolidation of the diagnostic effort required to test for various phenotypes important to the characterization of Salmonella. For example, drug resistance, serovar, virulence determinants, and other genome-based attributes can all be discerned using a genome sequence. Finally, genomic analysis, in conjunction with functional and phenotypic approaches, is beginning to provide new insights into the precise adaptive changes that permit persistence of NTS in so many diverse and challenging environmental niches.

A Generalized Potentiostat Adaptor for Multiplexed Electroanalysis.

Electrochemical measurements over an array of electrodes may be accomplished with one of three potentiostat architectures: a single-channel device which averages the signal from a number of interconnected electrodes, a multichannel device with dedicated circuits for each electrode, or a single-channel device with a multiplexer interface to isolate the signal from specific electrodes. Of these three architectures, the use of a multiplexer interface is best suited to facilitate measurements over individual electrodes without the need for large numbers of dedicated potentiostat channels. We present a versatile strategy for the development of flexible printed circuit (FPC) electrode arrays with accompanying multiplexing hardware to interface with single-channel potentiostats. The FPC array was fabricated with 78 individually addressable 0.3 mm diameter gold working electrodes and characterized using optical and scanning electron microscopy, energy dispersive spectroscopy, profilometry, impedance spectroscopy, and cyclic voltammetry to investigate the morphology, elemental composition, height profile, impedance characteristics, and electrochemical response, respectively. Interfacing the FPC array via a simple connector with three 32-channel ADG731 multiplexers permitted electrochemical measurements using single-channel commercial potentiostats. Voltammetric experiments were conducted to demonstrate the reliability, stability, and reproducibility of the FPC array and interfacing hardware. The combination of these devices represents an accessible hardware platform with robust, functionalizable electrodes, a simple connection interface with commercial potentiostats, and a low cost through the use of off-the-shelf components. Our reported strategy holds great promise to facilitate multiplexed electroanalysis in next-generation sensors to increase statistical sample size and multianalyte detection capabilities.

A clinical practice guideline using percentage of predicted forced vital capacity improves resource allocation for rib fracture patients.

BACKGROUND: Predicting rib fracture patients that will require higher-level care is a challenge during patient triage. Percentage of predicted forced vital capacity (FVC%) incorporates patient-specific factors to customize the measurements to each patient. A single institution transitioned from a clinical practice guideline (CPG) using absolute forced vital capacity (FVC) to one using FVC% to improve triage of rib fracture patients. This study compares the outcomes of patients before and after the CPG change. METHODS: A review of rib fracture patients was performed over a 3-year retrospective period (RETRO) and 1-year prospective period (PRO). RETRO patients were triaged by absolute FVC. Percentage of predicted FVC was used to triage PRO patients. Demographics, mechanism, Injury Severity Score, chest Abbreviated Injury Scale score, number of rib fractures, tube thoracostomy, intubation, admission to intensive care unit (ICU), transfer to ICU, hospital length of stay (LOS), ICU LOS, and mortality data were compared. A multivariable model was constructed to perform adjusted analysis for LOS. RESULTS: There were 588 patients eligible for the study, with 269 RETRO and 319 PRO patients. No significant differences in age, gender, or injury details were identified. Fewer tube thoracostomy were performed in PRO patients. Rates of intubation, admission to ICU, and mortality were similar. The PRO cohort had fewer ICU transfers and shorter LOS and ICU LOS. Multivariable logistic regression identified a 78% reduction in odds of ICU transfer among PRO patients. Adjusted analysis with multiple linear regression showed LOS was decreased 1.28 days by being a PRO patient in the study (B = -1.44; p < 0.001) with R2 = 0.198. CONCLUSION: Percentage of predicted FVC better stratified rib fracture patients leading to a decrease in transfers to the ICU, ICU LOS, and hospital LOS. By incorporating patient-specific factors into the triage decision, the new CPG optimized triage and decreased resource utilization over the study period. LEVEL OF EVIDENCE: Therapeutic/Care Management. Trauma, Rib, Triage, level IV.

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

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

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

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

Taxonomic and Functional Shifts in the Sprout Spent Irrigation Water Microbiome in Response to Salmonella Contamination of Alfalfa Seeds.

Despite recent advances in Salmonella-sprout research, little is known about the relationship between Salmonella and the sprout microbiome during sprouting. Sprout spent irrigation water (SSIW) provides an informative representation of the total microbiome of this primarily aquaponic crop. This study was designed to characterize the function and taxonomy of the most actively transcribed genes in SSIW from Salmonella enterica serovar Cubana-contaminated alfalfa seeds throughout the sprouting process. Genomic DNA and total RNA from SSIW was collected at regular intervals and sequenced using Illumina MiSeq and NextSeq platforms. Nucleic acid data were annotated using four different pipelines. Both metagenomic and metatranscriptomic analyses revealed a diverse and highly dynamic SSIW microbiome. A "core" SSIW microbiome comprised Klebsiella, Enterobacter, Pantoea, and Cronobacter The impact, however, of Salmonella contamination on alfalfa seeds influenced SSIW microbial community dynamics not only structurally but also functionally. Changes in genes associated with metabolism, genetic information processing, environmental information processing, and cellular processes were abundant and time dependent. At time points of 24 h, 48 h, and 96 h, totals of 541, 723, and 424 S Cubana genes, respectively, were transcribed at either higher or lower levels than at 0 h in SSIW during sprouting. An array of S Cubana genes (107) were induced at all three time points, including genes involved in biofilm formation and modulation, stress responses, and virulence and tolerance to antimicrobials. Taken together, these findings expand our understanding of the effect of Salmonella seed contamination on the sprout crop microbiome and metabolome.IMPORTANCE Interactions of human enteric pathogens like Salmonella with plants and plant microbiomes remain to be elucidated. The rapid development of next-generation sequencing technologies provides powerful tools enabling investigation of such interactions from broader and deeper perspectives. Using metagenomic and metatranscriptomic approaches, this study identified not only changes in microbiome structure of SSIW associated with sprouting but also changes in the gene expression patterns related to the sprouting process in response to Salmonella contamination of alfalfa seeds. This study advances our knowledge on Salmonella-plant (i.e., sprout) interaction.

Antimicrobial resistance and related gene analysis of Salmonella from egg and chicken sources by whole-genome sequencing.

Whole-genome sequencing (WGS) is a valuable tool in research on foodborne pathogens. In this study, a total of 143 isolates of Salmonella serotypes Enteritidis, Typhimurium, and Heidelberg sourced from eggs and chickens were analyzed for their antimicrobial resistance profiles using WGS data. The isolates carried high rate of genes resistant to aminoglycoside (70.63%), tetracycline (26.57%), fosfomycin (25.17%), sulfonamides (23.78%), and ß-lactamases (15.38%); and aadA was the most frequently observed antimicrobial resistance gene (ARG). Antimicrobial resistance varies by Salmonella serotypes, with Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis) isolates being highly resistant to aminoglycoside (particularly streptomycin); Salmonella ser. Typhimurium more resistant to aminoglycoside, tetracycline, and sulfonamides; and Salmonella ser. Heidelberg more resistant to aminoglycoside and fosfomycin. Salmonella ser. Typhimurium isolates presented more varieties of ARG than Salmonella ser. Enteritidis and Salmonella ser. Heidelberg. Our data showed that 5 isolates of Salmonella ser. Typhimurium and Salmonella ser. Heidelberg contained ARG resistant to ≥ 5 antimicrobials. In addition, 23 Salmonella isolates carried ARG resistant to 4 antimicrobials.

Food Safety Genomics and Connections to One Health and the Clinical Microbiology Laboratory.

This article describes the potential for one health surveillance of foodborne pathogens and disease using the revolutionary methodologies of whole genome sequencing. Whole genome sequencing of viral and bacterial pathogens is a natural fit to a one health perspective because these pathogens reside and are shared by humans, animals, and the environment and their genomes are compared easily regardless of where or from what host the pathogen was isolated. A genome provides a huge amount of data that can be analyzed for numerous applications. Sharing data coordinates surveillance efforts across the various disciplines.

Microevolution and Gain or Loss of Mobile Genetic Elements of Outbreak-Related Listeria monocytogenes in Food Processing Environments Identified by Whole Genome Sequencing Analysis.

Whole genome sequencing (WGS) analyses have been instrumental in traceback investigations of Listeria monocytogenes (Lm). To demonstrate how long-read sequencing analysis can capture and describe relationships among isolates from clinical, food, and environmental sources, we analyzed 366 long-read- and shotgun-sequenced isolates from 16 Lm outbreak strains associated with cantaloupe, leafy green, stone fruit, caramel apple, mung bean sprout, multiple cheese products, multiple ice cream products, and their production environments. The analyses demonstrated that outbreak strains could be distributed in different areas and zones of food production environments through persistent or repeated contamination. Multi-strain and multi-clone contamination were common. Further, WGS could differentiate among isolates collected at different time points or from different production lines in the same facility, revealing microevolution events in processing environments. Our comparison between complete and shotgun genomes showed that isolates of the same outbreak strain diversified mostly by gain/loss of plasmids and chromosome-borne prophages that constitute 2 to 5% of the chromosome. In contrast, other genes missing in the shotgun genomes were randomly scattered, constituting ~0.5% of the chromosome. Among different outbreak strains of the same CC, most gene-scale differences were due to gain/loss of mobile genetic elements, such as plasmids, chromosome-borne prophages, a Tn916 like transposon, and Listeria Genomic Island 2. The nucleotide variations in the same prophage and the same plasmid shared among isolates of the same outbreak strain were limited, which enabled different WGS tools to unambiguously cluster isolates of the same outbreak strain. In some outbreak strains, correlation between prophage gain/loss and single nucleotide polymorphism (SNP) accumulations in the genome backbone were observed.

Colonization and Internalization of Salmonella enterica and Its Prevalence in Cucumber Plants.

Consumption of cucumbers (Cucumis sativus var. sativus) has been linked to several foodborne outbreaks involving Salmonella enterica. The purpose of this work was to investigate the efficiency of colonization and internalization of S. enterica into cucumber plants by various routes of contamination. Produce-associated outbreak strains of Salmonella (a cocktail of serovars Javiana, Montevideo, Newport, Poona, and Typhimurium) were introduced to three cultivars of cucumber plants (two slicing cultivars and one pickling) via blossoms (ca. 6.4 log10 CFU/blossom, 4.5 log10 CFU/blossom, or 2.5 log10 CFU/blossom) or soil (ca. 8.3 log10 CFU/root zone) and were analyzed for prevalence of Salmonella contamination (internal and external) and serovar predominance in fruit and stems. Of the total slicing fruit harvested from Salmonella-inoculated blossoms (ca. 6.4, 4.5, or 2.5 log10 CFU/blossom), 83.9% (47/56), 81.4% (48/59) or 71.2% (84/118) were found colonized and 67.9% (38/56), 35.6% (21/59) or 22.0% (26/118) had Salmonella internalized into the fruit, respectively. S. Poona was the most prevalent serovar isolated on or in cucumber fruits at all inoculation levels. When soil was inoculated at 1 day post-transplant (dpt), 8% (10/120) of the plants were shown to translocate Salmonella to the lower stem 7 days post-inoculation (dpi). Results identified blossoms as an important route by which Salmonella internalized at a high percentage into cucumbers, and S. Poona, the same strain isolated from the 2015 outbreak of cucumbers imported from Mexico, was shown to be well-adapted to the blossom niche.

Genetic diversity and profiles of genes associated with virulence and stress resistance among isolates from the 2010-2013 interagency Listeria monocytogenes market basket survey.

Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010-2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon's diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events.

Temporal Dynamics of Salmonella enterica subsp. enterica Serovar Agona Isolates From a Recurrent Multistate Outbreak.

The largest outbreak of Salmonella Agona in the United States occurred in 1998. It affected more than 400 patients and was linked to toasted oat cereal. Ten years later, a similar outbreak occurred with the same outbreak strain linked to the same production facility. In this study, whole-genome sequence (WGS) data from a set of 46 Salmonella Agona including five isolates associated with the 1998 outbreak and 25 isolates associated with the 2008 outbreak were analyzed. From each outbreak one isolate was sequenced on the Pacific Biosciences RS II Sequencer to determine the complete genome sequence. We reconstructed a phylogenetic hypothesis of the samples using a reference-based method for identifying variable sites. Using Single Nucleotide Polymorphism (SNP) analyses, we were able to distinguish and separate Salmonella Agona isolates from both outbreaks with only a mean of eight SNP differences between them. The phylogeny illustrates that the 2008 outbreak involves direct descendants from the 1998 outbreak rather than a second independent contamination event. Based on these results, there is evidence supporting the persistence of Salmonella over time in food processing facilities and highlights the need for consistent monitoring and control of organisms in the supply chain to minimize the risk of successive outbreaks.