Benchmarking short and long read polishing tools for nanopore assemblies: achieving near-perfect genomes for outbreak isolates.

BACKGROUND: Oxford Nanopore provides high throughput sequencing platforms able to reconstruct complete bacterial genomes with 99.95% accuracy. However, even small levels of error can obscure the phylogenetic relationships between closely related isolates. Polishing tools have been developed to correct these errors, but it is uncertain if they obtain the accuracy needed for the high-resolution source tracking of foodborne illness outbreaks. RESULTS: We tested 132 combinations of assembly and short- and long-read polishing tools to assess their accuracy for reconstructing the genome sequences of 15 highly similar Salmonella enterica serovar Newport isolates from a 2020 onion outbreak. While long-read polishing alone improved accuracy, near perfect accuracy (99.9999% accuracy or ~ 5 nucleotide errors across the 4.8 Mbp genome, excluding low confidence regions) was only obtained by pipelines that combined both long- and short-read polishing tools. Notably, medaka was a more accurate and efficient long-read polisher than Racon. Among short-read polishers, NextPolish showed the highest accuracy, but Pilon, Polypolish, and POLCA performed similarly. Among the 5 best performing pipelines, polishing with medaka followed by NextPolish was the most common combination. Importantly, the order of polishing tools mattered i.e., using less accurate tools after more accurate ones introduced errors. Indels in homopolymers and repetitive regions, where the short reads could not be uniquely mapped, remained the most challenging errors to correct. CONCLUSIONS: Short reads are still needed to correct errors in nanopore sequenced assemblies to obtain the accuracy required for source tracking investigations. Our granular assessment of the performance of the polishing pipelines allowed us to suggest best practices for tool users and areas for improvement for tool developers.

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.

Prospective Genomic Surveillance Reveals Cryptic MRSA Outbreaks with Local to International Origins among NICU Patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause substantive morbidity and mortality in neonates. Using publicly available resources from the National Center of Biotechnology Information (NCBI) and Food and Drug Administration's (FDA) GalaxyTrakr pipeline, we illustrate the dynamics of MRSA colonization and infection in neonates. Over 217 days of prospective surveillance, analyses revealed concurrent MRSA transmission chains affecting 11 of 17 MRSA-colonized patients (65%), with two clusters that demonstrated intervals of more than a month among the appearance of isolates. All MRSA infected neonates (n = 3) showed previous colonization with the infecting strain. GalaxyTrakr clustering of the NICU strains, in the context of 21,521 international isolates deposited in NCBI's Pathogen Detection Resource, revealed NICU isolates to be distinct from adult MRSA strains seen locally and internationally. Clustering of the NICU strains within an international context enhanced the resolution of strain clusters and supported the rule-out of suspected, local transmission events within the NICU. Analyses also identified sequence type 1535 isolates, emergent in the Middle East, carrying a unique SCCmec with fusC and aac(6')-Ie/aph(2'')-1a that provided a multidrug-resistant phenotype. NICU genomic pathogen surveillance, leveraging public repositories and outbreak detection tools, supports rapid identification of cryptic MRSA clusters, and can inform infection prevention interventions for this vulnerable patient population. Results demonstrate that sporadic infections in the NICU may be indicative of hidden chains of asymptomatic transmission best identified with sequenced-based approaches.

Induction of the Viable-but-Nonculturable State in Salmonella Contaminating Dried Fruit.

Salmonella can become viable but nonculturable (VBNC) in response to environmental stressors, but the induction of the VBNC state in Salmonella contaminating ready-to-eat dried fruit is poorly characterized. Dried apples, strawberries, and raisins were mixed with a five-strain cocktail of Salmonella at 4% volume per weight of dried fruit at 109 CFU/g. The inoculated dried fruit were then dried in desiccators at 25°C until the water activity (aw) approximated that of the uninoculated dried fruit. However, Salmonella could not be recovered after drying, not even after enrichment, suggesting a population reduction of approximately 8 log CFU/g. To assess the potential impact of storage temperature on survival, dried apples were spot-inoculated with the Salmonella cocktail, dried under ambient atmosphere at 25°C, and stored at 4 and 25°C. Spot inoculation permitted recovery of Salmonella on dried apple after drying, with the population of Salmonella decreasing progressively on dried apples stored at 25°C until it was undetectable after about 46 days, even following enrichment. The population decline was noticeably slower at 4°C, with Salmonella being detected until 82 days. However, fluorescence microscopy and laser scanning confocal microscopy with the LIVE/DEAD BacLight bacterial viability system at time points at which no Salmonella could be recovered on growth media even following enrichment showed that a large proportion (56 to 85%) of the Salmonella cells on the dried fruit were viable. The data suggest that the unique combination of stressors in dried fruit can induce large numbers of VBNC cells of Salmonella. IMPORTANCE Salmonella is a leading foodborne pathogen globally causing numerous outbreaks of foodborne illnesses and remains the leading contributor to deaths attributed to foodborne disease in the United States and other industrialized nations. Therefore, efficient detection methods for Salmonella contaminating food are critical for public health and food safety. Culture-based microbiological methods are considered the gold standard for the detection and enumeration of Salmonella in food. Findings from this study suggest that unique stressors on dried fruit can induce the VBNC state in Salmonella, thus rendering it undetectable with culture-based methods even though the bacteria remain viable. Therefore, strong consideration should be given to using, in addition to culture-based methods, microscopic and molecular methods for the accurate detection of all viable and/or culturable cells of Salmonella contaminating dried fruit, as all of these cells have the potential to cause human illness.

Novel Cadmium Resistance Determinant in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that can cause severe disease (listeriosis) in susceptible individuals. It is ubiquitous in the environment and often exhibits resistance to heavy metals. One of the determinants that enables Listeria to tolerate exposure to cadmium is the cadAC efflux system, with CadA being a P-type ATPase. Three different cadA genes (designated cadA1 to cadA3) were previously characterized in L. monocytogenes A novel putative cadmium resistance gene (cadA4) was recently identified through whole-genome sequencing, but experimental confirmation for its involvement in cadmium resistance is lacking. In this study, we characterized cadA4 in L. monocytogenes strain F8027, a cadmium-resistant strain of serotype 4b. By screening a mariner-based transposon library of this strain, we identified a mutant with reduced tolerance to cadmium and that harbored a single transposon insertion in cadA4 The tolerance to cadmium was restored by genetic complementation with the cadmium resistance cassette (cadA4C), and enhanced cadmium tolerance was conferred to two unrelated cadmium-sensitive strains via heterologous complementation with cadA4C Cadmium exposure induced cadA4 expression, even at noninhibitory levels. Virulence assessments in the Galleria mellonella model suggested that a functional cadA4 suppressed virulence, potentially promoting commensal colonization of the insect larvae. Biofilm assays suggested that cadA4 inactivation reduced biofilm formation. These data not only confirm cadA4 as a novel cadmium resistance determinant in L. monocytogenes but also provide evidence for roles in virulence and biofilm formation.IMPORTANCEListeria monocytogenes is an intracellular foodborne pathogen causing the disease listeriosis, which is responsible for numerous hospitalizations and deaths every year. Among the adaptations that enable the survival of Listeria in the environment are the abilities to persist in biofilms, grow in the cold, and tolerate toxic compounds, such as heavy metals. Here, we characterized a novel determinant that was recently identified on a larger mobile genetic island through whole-genome sequencing. This gene (cadA4) was found to be responsible for cadmium detoxification and to be a divergent member of the Cad family of cadmium efflux pumps. Virulence assessments in a Galleria mellonella model suggested that cadA4 may suppress virulence. Additionally, cadA4 may be involved in the ability of Listeria to form biofilms. Beyond the role in cadmium detoxification, the involvement of cadA4 in other cellular functions potentially explains its retention and wide distribution in L. monocytogenes.

Identification of Novel Genes Mediating Survival of Salmonella on Low-Moisture Foods via Transposon Sequencing Analysis.

Salmonella enterica is the leading foodborne pathogen associated with outbreaks involving low-moisture foods (LMFs). However, the genes involved in Salmonella's long-term survival on LMFs remain poorly characterized. In this study, in-shell pistachios were inoculated with Tn5-based mutant libraries of S. Enteritidis P125109, S. Typhimurium 14028s, and S. Newport C4.2 at approximate 108 CFU/g and stored at 25°C. Transposon sequencing analysis (Tn-seq) was then employed to determine the relative abundance of each Tn5 insertion site immediately after inoculation (T0), after drying (T1), and at 120 days (T120). In S. Enteritidis, S. Typhimurium, and S. Newport mutant libraries, the relative abundance of 51, 80, and 101 Tn5 insertion sites, respectively, was significantly lower at T1 compared to T0, while in libraries of S. Enteritidis and S. Typhimurium the relative abundance of 42 and 68 Tn5 insertion sites, respectively, was significantly lower at T120 compared to T1. Tn5 insertion sites with reduced relative abundance in this competition assay were localized in DNA repair, lipopolysaccharide biosynthesis and stringent response genes. Twelve genes among those under strong negative selection in the competition assay were selected for further study. Whole gene deletion mutants in ten of these genes, sspA, barA, uvrB, damX, rfbD, uvrY, lrhA, yifE, rbsR, and ompR, were impaired for individual survival on pistachios. The findings highlight the value of combined mutagenesis and sequencing to identify novel genes important for the survival of Salmonella in low-moisture foods.

Validation of an ampicillin selection protocol to enrich for mutants of Listeria monocytogenes unable to replicate on fresh produce.

Several outbreaks of listeriosis have implicated fresh produce but genetic factors required for growth of Listeria monocytogenes on produce remain poorly characterized. Based on the fact that ß-lactam antibiotics only kill bacterial cells that are growing, we hypothesized that ampicillin selection can enrich for L. monocytogenes mutants unable to grow on produce. For validation, we examined relative recovery of L. monocytogenes strain 2011L-2858 and its cold-sensitive mutant L1E4 following inoculation of cantaloupe rind fragments with 1:1 mixture of the strains and incubation at 4°C with or without ampicillin. Listeria monocytogenes from rind fragments inoculated with the mixed cultures and incubated in the presence of ampicillin were used to inoculate fresh rind fragments for a second round of enrichment. In the presence of ampicillin, the proportion of L1E4 increased from 55% on day 0 to 78% on day 14, with higher recovery (85% after 14 days) in the second round of enrichment. These data suggested that L1E4 was enriched on cantaloupe rind fragments while growing cells of the wildtype were killed by ampicillin. Application of this protocol to transposon mutant libraries from three L. monocytogenes strains yielded several mutants unable to grow on cantaloupe. Thus, ampicillin selection can facilitate discovery of genes essential for growth of L. monocytogenes on fresh produce.

Predicting the Growth of Listeria monocytogenes and Salmonella Typhimurium in Diced Celery, Onions, and Tomatoes during Simulated Commercial Transport, Retail Storage, and Display.

Temperature is arguably the most important factor affecting microbial proliferation in fresh-cut produce. In this study, growth of Listeria monocytogenes in diced onions and celery and Salmonella Typhimurium in diced tomatoes was determined in modified atmosphere packages and snap-fit containers using three fluctuating temperature scenarios for transport, retail storage, and display. As expected, L. monocytogenes growth in diced onions and celery varied depending on the extent of temperature abuse, with exposure to high and intermediate temperature-abuse scenarios generally being growth supportive. A Baranyi primary model with a square-root secondary model for maximum growth rate, and a linear model for maximum population density, were used to estimate Listeria growth under fluctuating temperature. Accuracy and acceptability of the model prediction were evaluated in terms of root mean square error (RMSE) and acceptable prediction zone (APZ), respectively. Overall, growth predictions for L. monocytogenes were more accurate for celery (RMSE, 0.28 to 0.47) than onions (RMSE, 0.42 to 1.53) under the fluctuating temperature scenarios tested. However, both predictions yielded APZ values that ranged from 82 to 100% for celery and 36 to 78% for onions. In contrast, Salmonella Typhimurium populations increased more than 1 log CFU/g in diced tomatoes under the three fluctuating temperature scenarios studied. Overall, these diced products packaged under a high-oxygen atmosphere showed decreased pathogen growth compared with product stored in a passive modified atmosphere. Findings from this study will be particularly useful in assessing the risk associated with consumption of diced celery, tomatoes, and onions and in designing effective packaging strategies to minimize pathogen growth in fresh-cut produce.

The Listeria monocytogenes Key Virulence Determinants hly and prfA are involved in Biofilm Formation and Aggregation but not Colonization of Fresh Produce.

Listeria monocytogenes has been extensively studied as a model facultative intracellular pathogen. While the roles of major virulence factors in host-pathogen interactions have been extensively characterized, recent work suggests that some of these factors can also contribute to environmental proliferation of this pathogen. In this study, we characterized two non-hemolytic transposon mutants of strain 2011L-2858 (serotype 1/2b), implicated in the 2011 listeriosis outbreak via whole cantaloupe, for their capacity to form biofilms on polystyrene, aggregate, and colonize cantaloupe rind. One mutant harbored a single mariner-based transposon insertion in hly, encoding the hemolysin Listeriolysin O, while the other harbored a single insertion in prfA, encoding PrfA, a master regulator for hly and numerous other virulence genes. Biofilm formation was significantly reduced in the prfA mutant, and to a lesser extent, in the hly mutant. Inactivation of either hly or prfA significantly reduced L. monocytogenes aggregation. However, both mutants adhered similarly to the wildtype parental strain on cantaloupe rind at either 25 or 37°C. Furthermore, growth and competitive fitness of the mutants on cantaloupe rind was not significantly impacted at either temperature. The findings suggest that, in spite of their involvement in biofilm formation and aggregation, these key virulence determinants may not be required for the ability of L. monocytogenes to colonize fresh produce.

Capacity of Listeria monocytogenes Strains from the 2011 Cantaloupe Outbreak To Adhere, Survive, and Grow on Cantaloupe.

The 2011 listeriosis outbreak attributed to whole cantaloupe involved several genetically distinct strains of serotypes 1/2a and 1/2b that had not been previously reported in invasive listeriosis outbreaks. Here we investigated the potential of strains from the 2011 cantaloupe outbreak to adhere, survive, and grow on cantaloupe rind and flesh and in juice extracted from cantaloupe at different temperatures (4, 8, and 25°C). All strains were able to adhere and grow, with ∼10-fold increases after 7 days at 4 or 8°C and after 24 h at 25°C, with a propensity for more growth on rind than on flesh or in extract. No significant differences in growth potential were noted among the different strains or between them and unrelated strains from other listeriosis outbreaks involving celery, deli meats, or hot dogs. Similarly to the cantaloupe outbreak strains, these other strains exhibited greater propensity for growth on rind than on flesh or in extract. Rinsing of cantaloupe fragments in sterile water resulted in temporary reductions of the populations by 50- to 100-fold, suggesting the potential of such washing to reduce risk if the produce is promptly consumed. The absence of marked differences in adherence or growth between the cantaloupe outbreak strains and strains from other outbreaks highlights the need to further characterize the 2011 cantaloupe outbreak strains and elucidate potential biological attributes that contributed to their implication in the outbreak.