Salmonella Genomics and Population Analyses Reveal High Inter- and Intraserovar Diversity in Freshwater.

Freshwater can support the survival of the enteric pathogen Salmonella, though temporal Salmonella diversity in a large watershed has not been assessed. At 28 locations within the Susquehanna River basin, 10-liter samples were assessed in spring and summer over 2 years. Salmonella prevalence was 49%, and increased river discharge was the main driver of Salmonella presence. The amplicon-based sequencing tool, CRISPR-SeroSeq, was used to determine serovar population diversity and detected 25 different Salmonella serovars, including up to 10 serovars from a single water sample. On average, there were three serovars per sample, and 80% of Salmonella-positive samples contained more than one serovar. Serovars Give, Typhimurium, Thompson, and Infantis were identified throughout the watershed and over multiple collections. Seasonal differences were evident: serovar Give was abundant in the spring, whereas serovar Infantis was more frequently identified in the summer. Eight of the ten serovars most commonly associated with human illness were detected in this study. Crucially, six of these serovars often existed in the background, where they were masked by a more abundant serovar(s) in a sample. Serovars Enteritidis and Typhimurium, especially, were masked in 71 and 78% of samples where they were detected, respectively. Whole-genome sequencing-based phylogeny demonstrated that strains within the same serovar collected throughout the watershed were also very diverse. The Susquehanna River basin is the largest system where Salmonella prevalence and serovar diversity have been temporally and spatially investigated, and this study reveals an extraordinary level of inter- and intraserovar diversity.IMPORTANCESalmonella is a leading cause of bacterial foodborne illness in the United States, and outbreaks linked to fresh produce are increasing. Understanding Salmonella ecology in freshwater is of importance, especially where irrigation practices or recreational use occur. As the third largest river in the United States east of the Mississippi, the Susquehanna River is the largest freshwater contributor to the Chesapeake Bay, and it is the largest river system where Salmonella diversity has been studied. Rainfall and subsequent high river discharge rates were the greatest indicators of Salmonella presence in the Susquehanna and its tributaries. Several Salmonella serovars were identified, including eight commonly associated with foodborne illness. Many clinically important serovars were present at a low frequency within individual samples and so could not be detected by conventional culture methods. The technologies employed here reveal an average of three serovars in a 10-liter sample of water and up to 10 serovars in a single sample.

Salmonella enterica colonization and fitness in pre-harvest cantaloupe production.

Cantaloupes have emerged as significant vehicles of widespread foodborne illness outbreaks caused by bacterial pathogens, including Salmonella. The purpose of this study was to investigate the efficiency of Salmonella colonization and internalization in cantaloupes by relevant routes of contamination. Cantaloupe plants (Cucumis melo 'reticulatus') from two cultivars 'Athena' (Eastern) and 'Primo' (Western) were grown from commercial seed. Plants were maintained in the NCSU BSL-3P phytotron greenhouse. Salmonella enterica (a cocktail of cantaloupe-associated outbreak serovars Javiana, Newport, Panama, Poona and Typhimurium) contamination was introduced via blossoms or soil at ca. 4.4 log10 CFU/blossom or 8.4 log10 CFU/root zone, respectively. Cantaloupes were analyzed for Salmonella by enrichment in accordance with modified FDA-BAM methods. Five randomly chosen colonies from each Salmonella-positive sample were typed using the Agilent 2100 bioanalyzer following multiplex PCR. Data were analyzed for prevalence of contamination and serovar predominance in fruit, stems and soil. Of the total cantaloupe fruit harvested from Salmonella-inoculated blossoms (n = 63), 89% (56/63) were externally contaminated and 73% (46/63) had Salmonella internalized into the fruit. Serovar Panama was the most commonly isolated from the surface of fruit while S. Panama and S. Poona were the most prevalent inside the fruit. When soil was inoculated with Salmonella at one day post-transplant, 13% (8/60) of the plants were shown to translocate the organism to the lower stem (ca. 4 cm) by 7 days post-inoculation (dpi). We observed Salmonella persistence in the soil up to 60 dpi with S. Newport being the predominant serovar at 10 and 20 dpi. These data demonstrate that contaminated soil and blossoms can lead to Salmonella internalization into the plant or fruit at a relatively high frequency.

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.

Evaluation and comparison of rapid methods for the detection of Salmonella in naturally contaminated pine nuts using different pre enrichment media.

Foodborne outbreaks, involving pine nuts and peanut butter, illustrate the need to rapidly detect Salmonella in low moisture foods. However, the current Bacteriological Analytical Manual (BAM) culture method for Salmonella, using lactose broth (LB) as a pre enrichment medium, has not reliably supported real-time quantitative PCR (qPCR) assays for certain foods. We evaluated two qPCR assays in LB and four other pre enrichment media: buffered peptone water (BPW), modified BPW (mBPW), Universal Pre enrichment broth (UPB), and BAX(®) MP media to detect Salmonella in naturally-contaminated pine nuts (2011 outbreak). A four-way comparison among culture method, Pathatrix(®) Auto, VIDAS(®) Easy SLM, and qPCR was conducted. Automated DNA extraction techniques were compared with manual extraction methods (boiling or InstaGene™). There were no significant differences (P > 0.05) among the five pre enrichment media for pine nuts using the culture method. While both qPCR assays produced significantly (P ≤ 0.05) higher false negatives in 24 h pre enriched LB than in the other four media, they were as sensitive as the culture method in BPW, mBPW, UPB, and BAX media. The VIDAS Easy and qPCR were equivalent; Pathatrix was the least effective method. The Automatic PrepSEQ™ DNA extraction, using 1000 µL of pre enrichment, was as effective as manual extraction methods.

Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022.

Background: Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobial-resistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods: We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings: The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial-susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended-spectrum ß-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation: Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first-line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent. Funding: Partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services as part of an award, FDU001818, with 30% percent funded by FDA/HHS; and by Agencia de Investigación y Desarrollo de Chile (ANID) through FONDECYT de Postdoctorado Folio 3230796 and Folio 3210317, FONDECYT Regular Folio 1231082, and ANID-Millennium Science Initiative Program-ICN2021_044.

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.

bettercallsal: better calling of Salmonella serotypes from enrichment cultures using shotgun metagenomic profiling and its application in an outbreak setting.

Most current Salmonella subtyping analyses rely on whole genome sequencing (WGS), which focuses on the high-resolution analysis of single genomes or multiple single genomes from the isolated colonies on microbiological agar plates. In this study, we introduce bioinformatics innovations for a metagenomic outbreak response workflow that accurately identifies multiple Salmonella serovars at the same time. bettercallsal is one of the first analysis tools to identify multiple Salmonella enterica serotypes from metagenomic or quasi-metagenomic datasets with high accuracy, allowing these isolate-independent methods to be incorporated into surveillance and root cause investigations. It was tested on an in silico benchmark dataset comprising 29 unique Salmonella serovars, 46 non-Salmonella bacterial genomes, and 10 viral genomes at varying read depths and on previously well-characterized and sequenced non-selective primary and selective enrichments of papaya and peach samples from separate outbreak investigations that resulted in the identification of multiple Salmonella serovars using traditional isolate culturing and WGS as well as nucleic acid assays. Analyses were also conducted on these datasets using a custom-built k-mer tool, SeqSero2, and Kallisto to compare serotype calling to bettercallsal. The in silico dataset analyzed with bettercallsal achieved the maximum precision, recall, and accuracy of 100, 83, and 94%, respectively. In the papaya outbreak samples, bettercallsal identified the presence of multiple serovars in agreement with the Luminex® xMAP assay results and also identified more serovars per sample, as evidenced by NCBI SNP clustering. In peach outbreak samples, bettercallsal identified two serovars in concordance with k-mer analysis and the Luminex xMAP assay. The genome hit reported by bettercallsal clustered with the chicken isolate genome, as reported by the FDA peach outbreak investigation from sequenced isolates (WGS). Overall, bettercallsal outperformed k-mer, Seqsero2, and Kallisto in identifying multiple serovars from enrichment cultures using shotgun metagenomic sequencing.

An Outbreak Investigation of Salmonella Typhimurium Illnesses in the United States Linked to Packaged Leafy Greens Produced at a Controlled Environment Agriculture Indoor Hydroponic Operation - 2021.

In 2021, the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and state partners investigated a multistate outbreak of Salmonella Typhimurium illnesses linked to packaged leafy greens from a controlled environment agriculture (CEA) operation in Illinois. Thirty-one illnesses and four hospitalizations were reported in four states, with a significant epidemiologic signal for packaged leafy greens from Farm A. A traceback investigation for leafy greens included seven points of service (POS) with food exposure data from eight ill people. Each POS was supplied leafy greens by Farm A. FDA investigators observed operations at Farm A and noted that 1) the firm did not consider their indoor hydroponic pond water as agricultural water, 2) condensate dripping from the chiller water supply line inside the building, and 3) unprotected outdoor storage of packaged soilless growth media and pallets used for finished product. FDA collected 25 product, water, and environmental samples from Farm A. The outbreak strain was recovered from a water sample collected from a stormwater drainage basin located on the property adjacent to Farm A. In addition, an isolate of Salmonella Liverpool was recovered from two indoor growing ponds within the same growing house, but no illnesses were linked to the isolate. Farm A voluntarily recalled all implicated products and provided their root cause analysis (RCA) and return-to-market plan to FDA. While the source and route of the contamination were not determined by the RCA, epidemiologic and traceback evidence confirmed the packaged salads consumed by ill persons were produced by Farm A. This was the first investigation of a multistate foodborne illness outbreak associated with leafy greens grown in a CEA operation. This outbreak demonstrated the need for growers using hydroponic methods to review their practices for potential sources and routes of contamination and to reduce food safety risks when identified.

Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-electrospray ionization mass spectrometry: isolate testing and analysis of food samples.

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.

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.

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.

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production.

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012-2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech's Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate - TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields.

Recent and emerging innovations in Salmonella detection: a food and environmental perspective.

Salmonella is a diverse genus of Gram-negative bacilli and a major foodborne pathogen responsible for more than a million illnesses annually in the United States alone. Rapid, reliable detection and identification of this pathogen in food and environmental sources is key to safeguarding the food supply. Traditional microbiological culture techniques have been the 'gold standard' for State and Federal regulators. Unfortunately, the time to result is too long to effectively monitor foodstuffs, especially those with very short shelf lives. Advances in traditional microbiology and molecular biology over the past 25 years have greatly improved the speed at which this pathogen is detected. Nonetheless, food and environmental samples possess a distinctive set of challenges for these newer, more rapid methodologies. Furthermore, more detailed identification and subtyping strategies still rely heavily on the availability of a pure isolate. However, major shifts in DNA sequencing technologies are meeting this challenge by advancing the detection, identification and subtyping of Salmonella towards a culture-independent diagnostic framework. This review will focus on current approaches and state-of-the-art next-generation advances in the detection, identification and subtyping of Salmonella from food and environmental sources.

Ecological prevalence, genetic diversity, and epidemiological aspects of Salmonella isolated from tomato agricultural regions of the Virginia Eastern Shore.

Virginia is the third largest producer of fresh-market tomatoes in the United States. Tomatoes grown along the eastern shore of Virginia are implicated almost yearly in Salmonella illnesses. Traceback implicates contamination occurring in the pre-harvest environment. To get a better understanding of the ecological niches of Salmonella in the tomato agricultural environment, a 2-year study was undertaken at a regional agricultural research farm in Virginia. Environmental samples, including tomato (fruit, blossoms, and leaves), irrigation water, surface water and sediment, were collected over the growing season. These samples were analyzed for the presence of Salmonella using modified FDA-BAM methods. Molecular assays were used to screen the samples. Over 1500 samples were tested. Seventy-five samples tested positive for Salmonella yielding over 230 isolates. The most commonly isolated serovars were S. Newport and S. Javiana with pulsed-field gel electrophoresis yielding 39 different patterns. Genetic diversity was further underscored among many other serotypes, which showed multiple PFGE subtypes. Whole genome sequencing (WGS) of several S. Newport isolates collected in 2010 compared to clinical isolates associated with tomato consumption showed very few single nucleotide differences between environmental isolates and clinical isolates suggesting a source link to Salmonella contaminated tomatoes. Nearly all isolates collected during two growing seasons of surveillance were obtained from surface water and sediment sources pointing to these sites as long-term reservoirs for persistent and endemic contamination of this environment.

Phylogenetic evaluation of the 'Typhimurium' complex of Salmonella strains using a seven-gene multi-locus sequence analysis.

Salmonella enterica comprises over 2500 serovars, many of which are significant foodborne pathogens in humans. The ability to subtype these microbes is difficult due to the highly clonal nature of many Salmonella strains and a lack of congruence among traditional typing approaches. This work examines the phylogenetic utility of a multi-locus sequence typing (MLST) approach to discriminate between members of a closely related collection of salmonellae, the Salmonella reference collection A (SARA). This 72 strain collection, referred to as the 'Typhimurium' complex, consists of S. Typhimurium and its four closest serological relatives. In this analysis, nucleotide sequences from seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were PCR amplified, sequenced, and combined into a single concatenated character matrix providing 3360bp for cladistic analysis. The resultant most parsimonious tree yielded seven clades of Salmonella strains that partitioned largely along serovar divisions within the collection except for five 'Paratyphi B' strains, two 'Saintpaul' strains, and two 'Typhimurium' strains. Convergence in the SARA tree was approximately 20% indicating that the vast majority of sequence changes were phylogenetically informative. Despite a high consistency among nucleotide substitutions, analysis of congruence identified several SARA strains with recombinant alleles in the concatenated matrix. These findings point to important differences among phylogenetic contributions made by the individual genes comprising this MLST dataset.