Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Characterizing the Genetic Diversity of Salmonella Isolated from U.S. Raw Inshell Pistachios Using Whole Genome Sequencing.

The genetic diversity of 169 Salmonella isolates from pistachios collected from California storage silos during the 2010, 2011, and 2012 harvests (silo survey isolates) was determined by analyzing the whole genome sequence data using the CFSAN SNP pipeline developed by the U.S. Food and Drug Administration's Center for Food Safety and Applied Nutrition. Salmonella isolates clustered by serovars Agona, Enteritidis, Montevideo, Sandiego, Senftenberg, Liverpool, Tennessee, and Worthington in the phylogenetic tree. Within each serovar, isolates grouped into one or two clusters (≤14 SNPs). Two distinct clusters (>14 SNPs; A and B) were identified for Salmonella Enteritidis, Montevideo, and Liverpool for a total of 11 unique strains. Sequences of representative silo survey isolates clustered with sequences of Salmonella strains isolated from U.S. pistachio-associated samples collected between 2008 and 2018 available on the National Center for Biotechnology Information database, and, in all but two cases, not with sequences of Salmonella strains recovered from raw California almonds from 2001 through 2013. The genomic evidence suggests that strains of Salmonella Agona, Liverpool Cluster A, Montevideo Clusters A and B, Senftenberg, and Worthington have persisted in the California pistachio environment for ≥3 years and some of these strains have been reported exclusively in association with pistachios.

Impact of irrigation water quality on human norovirus surrogate survival during leafy green production.

Introduction: The impact of water quality on the survival of human norovirus (NoV) was determined in irrigation water field run-off (tail water) and well water from a representative Central Coast vegetable production site in the Salinas Valley, California. Methods: Tail water, well water, and ultrapure water samples were inoculated separately with two surrogate viruses for human NoV-Tulane virus (TV) and murine norovirus (MNV)-to achieve a titer of 1×105 plaque forming units (PFU)/ml. Samples were stored at 11, 19, and 24°C for 28 days. Additionally, inoculated water was applied to soil collected from a vegetable production site in the Salinas Valley or to the surface of growing romaine lettuce leaves, and virus infectivity was evaluated for 28 days in a growth chamber. Results: Virus survival was similar for water stored at 11, 19, and 24°C and there was no difference in infectivity based on water quality. After 28 days, a maximum 1.5 log reduction was observed for both TV and MNV. TV decreased by 1.97-2.26 log and MNV decreased by 1.28- 1.48 logs after 28 days in soil; infectivity was not influenced by water type. Infectious TV and MNV were recovered from lettuce surfaces for up to 7 and 10 days after inoculation, respectively. Across the experiments there was no significant impact of water quality on the stability of the human NoV surrogates. Discussion: Overall, the human NoV surrogates were highly stable in water with a less than 1.5 log reduction over 28 days and no difference observed based on the water quality. In soil, the titer of TV declined by approximately 2 logs over 28 days, while MNV declined by 1 log during the same time interval, suggesting surrogate-specific inactivation dynamics in the soil tested in this study. A 5-log reduction in MNV (day 10 post inoculation) and TV (day 14 post inoculation) was observed on lettuce leaves, and the inactivation kinetics were not significantly impacted by the quality of water used. These results suggest that human NoV would be highly stable in water, and the quality of the water (e.g., nutrient content, salinity, and turbidity) does not significantly impact viral infectivity.

Water Application Method Influences Survival or Growth of Escherichia coli on Bulb Onions during Field Curing.

ABSTRACT: The impact of water application method on bacterial survival at or after the final irrigation was evaluated in bulb onions during commercially relevant field drying (curing). A three-strain rifampin-resistant cocktail of Escherichia coli was introduced to onions via a single overhead spray application in two separate trials (5.22 [trial 1] or 2.40 [trial 2] log CFU per onion) 2 to 3 days after the final irrigation. Onions were lifted from the soil 8 days after spray inoculation and, in some cases, foliage was removed (topping); onions remained in the field for an additional ca. 2 weeks (total ca. 3 weeks of curing). E. coli populations declined on the onions in the first 4 h after spray inoculation. E. coli was recovered from 38 (48%) or 28 (35%) of 80 whole-onion enrichments at the end of curing in trials 1 or 2, respectively. Topping did not significantly impact the percentage of E. coli-positive onions detected at the end of curing. From 8 h to 21 days, E. coli populations on positive onions ranged from 1 CFU per onion to 7 log CFU per onion in both trials, representing a potential risk of E. coli growth with overhead application of contaminated water at the end of onion production. In trial 2, additional rows of onions were inoculated via a 22-cm subsurface or surface drip irrigation line (1.94 log CFU/mL for 2.5 h). E. coli was detected in 0 (subsurface) and 4 (surface) of 50 whole-onion enrichments 3 h after the initiation of drip irrigation. Positive onions were detected at days 1 (4 of 50) and 7 (1 of 50) with subsurface drip inoculation, and at days 1 (7 of 50), 7 (2 of 50), and 14 (2 of 50) with surface drip inoculation. E. coli was not detected in whole-onion enrichments at the end of curing when inoculated by subsurface (0 of 50) or surface (0 of 50) drip irrigation. Application of contaminated water through drip irrigation, when coupled with field curing, results in low rates of contamination of bulb onions at the time of harvest.

Evaluation of glove type on survival and transfer of Escherichia coli in model systems and during hand harvesting of lettuce.

Background: Both reusable and single-use gloves can be employed during hand harvesting of lettuce and leafy greens. The impact of glove type on survival and transfer of Escherichia coli was evaluated using agar or lettuce in a laboratory setting and during simulated lettuce harvesting in the field. Results: Textured and smooth reusable latex and smooth disposable latex gloves inoculated with E. coli were sequentially touched to 10 or 20 agar plates or 20 lettuce leaves (n = 6; laboratory) or used to sequentially harvest 20 heads of lettuce (n = 6; field). E. coli was recovered by enrichment from significantly fewer leaves (46%; 55 of 120) or heads (26%; 31 of 120) of lettuce when inoculated reusable textured gloves were used compared with disposable gloves (leaves: 98%; 118 of 120, or heads: 74%; 89 of 120). In contrast, when a single head of lettuce was the point source for glove contamination, there was no significant difference in the number of E. coli-positive lettuce heads harvested with reusable textured (71%; 85 of 120) or disposable gloves (75%; 90 of 120). In either field-contamination scenario, at the 20th head of lettuce harvested with a single glove (final sample point), E. coli was recovered from one to five of six lettuce heads across experimental trials. Conclusion: Contamination of a glove from a single point source can lead to subsequent contamination of multiple heads of lettuce during hand harvesting, showing the importance of policies to manage hand hygiene and glove use for harvest crews.

Conditions at the time of inoculation influence survival of attenuated Escherichia coli O157:H7 on field-inoculated lettuce.

The impact of plant development, environmental conditions at the time of inoculation, and inoculum concentration on survival of attenuated BSL1 Escherichia coli O157:H7 strain ATCC 700728 on field-grown romaine lettuce was evaluated over 3 years. E. coli 700728 was inoculated onto 4- and 6-week-old romaine lettuce plants in the Salinas Valley, CA, at night or the next morning with either low (5 log) or high (7 log) cell numbers per plant to simulate a single aqueous contamination event. At night, when leaf wetness and humidity levels were high, E. coli cell numbers declined by 0.5 log CFU/plant over the first 8-10 h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2 h. However, similar numbers of E. coli were retrieved from lettuce plants at 2 and 7 days. E. coli cell numbers per plant were significantly lower (P < 0.05) 7 days after application onto 4-week-old compared to 6-week-old plants. E. coli 700728 could be recovered by plating or enrichment from a greater proportion of plants for longer times when inoculated at high compared with low initial concentrations and after inoculation of 6-week-old plants compared with 4-week-old plants, even at the low initial inoculum. A contamination event near harvest or when leaf wetness and humidity levels are high may enhance survivability, even when low numbers of E. coli are introduced.

RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants.

The capacity to distinguish between living and dead cells is an important, but often unrealized, attribute of rapid detection methods for foodborne pathogens. In this study, the numbers of enterohemorrhagic Escherichia coli O157:H7 after inoculation onto Romaine lettuce plants and on plastic (abiotic) surfaces were measured over time by culturing, and quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, and reverse transcriptase (RT)-qPCR targeting E. coli O157:H7 gapA, rfbE, eae, and lpfA genes and gene transcripts. On Romaine lettuce plants incubated at low relative humidity, E. coli O157:H7 cell numbers declined 10(7)-fold within 96 h according to culture-based assessments. In contrast, there were no reductions in E. coli levels according to qPCR and only 100- and 1000-fold lower numbers per leaf by RT-qPCR and PMA-qPCR, respectively. Similar results were obtained upon exposure of E. coli O157:H7 to desiccation conditions on a sterile plastic surface. Subsequent investigation of mixtures of living and dead E. coli O157:H7 cells strongly indicated that PMA-qPCR detection was subject to false-positive enumerations of viable targets when in the presence of 100-fold higher numbers of dead cells. RT-qPCR measurements of killed E. coli O157:H7 as well as for RNaseA-treated E. coli RNA confirmed that transcripts from dead cells and highly degraded RNA were also amplified by RT-qPCR. These findings show that neither PMA-qPCR nor RT-qPCR provide accurate estimates of bacterial viability in environments where growth and survival is limited.

Evaluation of different approaches for modeling Escherichia coli O157:H7 survival on field lettuce.

The ability to predict the behavior of Escherichia coli O157:H7 on contaminated field lettuce is essential for the development of accurate quantitative microbial risk assessments. The survival pattern of the species was assessed from several data sets derived from field-based experiments, which were analyzed by regression analysis fitting one monophasic model (log-linear) and two biphasic (Weibull and Cerf's model) models. Probabilistic models were also simulated with @RISK™, integrating the fitted monophasic and biphasic models in order to analyze their impact on the estimate of the extent of die-off subsequent to a contamination event in the field. Regression analysis indicated that E. coli O157:H7 followed a biphasic decay pattern in most cases, with the Weibull and Cerf's model showing similar good fit to individual and pooled survival data. Furthermore, results from the stochastic analysis demonstrated that using the log-linear model could lead to different risk estimates from those obtained with biphasic models, with a lower prevalence in the former scenario as no tailing is assumed in this model. The models and results derived from this work provide the first suitable mathematical base upon which to build probabilistic models to predict the fate of E. coli O157:H7 on field-grown leafy green vegetable.

Assessments of total and viable Escherichia coli O157:H7 on field and laboratory grown lettuce.

Leafy green produce has been associated with numerous outbreaks of foodborne illness caused by strains of Escherichia coli O157:H7. While the amounts of culturable E. coli O157:H7 rapidly decline after introduction onto lettuce in the field, it remains to be determined whether the reduction in cell numbers is due to losses in cell viability, cell injury and a subsequent inability to be detected by standard laboratory culturing methods, or a lack of adherence and hence rapid removal of the organism from the plants during application. To assess which of these options is most relevant for E. coli O157:H7 on leafy green produce, we developed and applied a propidium monoazide (PMA) real-time PCR assay to quantify viable (with PMA) and total (without PMA) E. coli O157:H7 cells on growth chamber and field-grown lettuce. E. coli O157:H7, suspended in 0.1% peptone, was inoculated onto 4-week-old lettuce plants at a level of approximately 10(6) CFU/plant. In the growth chamber at low relative humidity (30%), culturable amounts of the nontoxigenic E. coli O157:H7 strain ATCC 700728 and the virulent strain EC4045 declined 100 to 1000-fold in 24 h. Fewer E. coli O157:H7 cells survived when applied onto plants in droplets with a pipette compared with a fine spray inoculation. Total cells for both strains were equivalent to inoculum levels for 7 days after application, and viable cell quantities determined by PMA real-time PCR were approximately 10(4) greater than found by colony enumeration. Within 2 h after application onto plants in the field, the number of culturable E. coli ATCC 700728 was reduced by up to 1000-fold, whereas PCR-based assessments showed that total cell amounts were equivalent to inoculum levels. These findings show that shortly after inoculation onto plants, the majority of E. coli O157:H7 cells either die or are no longer culturable.

Season, irrigation, leaf age, and Escherichia coli inoculation influence the bacterial diversity in the lettuce phyllosphere.

The developmental and temporal succession patterns and disturbance responses of phyllosphere bacterial communities are largely unknown. These factors might influence the capacity of human pathogens to persist in association with those communities on agriculturally-relevant plants. In this study, the phyllosphere microbiota was identified for Romaine lettuce plants grown in the Salinas Valley, CA, USA from four plantings performed over 2 years and including two irrigation methods and inoculations with an attenuated strain of Escherichia coli O157:H7. High-throughput DNA pyrosequencing of the V5 to V9 variable regions of bacterial 16S rRNA genes recovered in lettuce leaf washes revealed that the bacterial diversity in the phyllosphere was distinct for each field trial but was also strongly correlated with the season of planting. Firmicutes were generally most abundant in early season (June) plantings and Proteobacteria comprised the majority of bacteria recovered later in the year (August and October). Comparisons within individual field trials showed that bacterial diversity differed between sprinkler (overhead) and drip (surface) irrigated lettuce and increased over time as the plants grew. The microbiota were also distinct between control and E. coli O157:H7-inoculated plants and between E. coli O157:H7-inoculated plants with and without surviving pathogen cells. The bacterial inhabitants of the phyllosphere therefore appear to be affected by seasonal, irrigation, and biological factors in ways that are relevant for assessments of fresh produce food safety.

Fate of Escherichia coli O157:H7 in field-inoculated lettuce.

Impact of drip and overhead sprinkler irrigation on the persistence of attenuated Escherichia coli O157:H7 in the lettuce phyllosphere was investigated using a split-plot design in four field trials conducted in the Salinas Valley, California, between summer 2007 and fall 2009. Rifampicin-resistant attenuated E. coli O157:H7 ATCC 700728 (BLS1) was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old lettuce plant foliage with a spray bottle at a level of 7 log CFU ml⁻¹. When E. coli O157:H7 was inoculated onto 2-week-old plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation, respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7 declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks post-inoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on lettuce.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

A frameshift in the coding region of a novel tomato class I basic chitinase gene makes it a pseudogene with a functional wound-responsive promoter.

A putative class I basic chitinase gene, assigned as psiBCH, was cloned from a tomato breeding line NC 24E. The gene contains a coding region with two introns. The predicted psiBCH open reading frame (ORF) is 971 bp and exhibits 81-88% identity at the nucleotide level with known class I basic chitinase genes from the Solanaceae family. However, the presence of a stop codon caused by a frameshift in the ORF of psiBCH makes it unusual among the other class I plant basic chitinases. This stop codon might be involved in the lower accumulation of fully spliced psiBCH RNA caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. Sequence analysis of the 1883-bp 5'-flanking region of the psiBCH gene revealed the presence of potential wound-response promoter elements. To study the transcriptional regulation of the psiBCH gene, its 5'-flanking region containing the putative promoter was fused to the gus reporter gene and introduced into the tobacco genome via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were functionally assayed for beta-glucuronidase activity. The psiBCH promoter drives the reporter gene expression in response to wounding stimuli. psiBCH promoter-GUS analysis indicates that wound-response of the tobacco transgene was rapid and localized in the wounded area following mechanical wounding. Therefore, our results suggest that the psiBCH promoter can provide targeted expression of genes, such as protease inhibitors in response to pest attack.

Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D.

Bacillus subtilis AU195 produces bacillomycin D, a cyclic lipopeptide that is an inhibitor of the aflatoxin producing fungus Aspergillus flavus. Sequence analysis of the bacillomycin D operon revealed four ORFs with the structural organization of the peptide synthetases. Disruption of ORF 2, which links the amino acid moiety to the b-amino fatty acid, resulted in the loss of antifungal activity. By comparing the sequence of bacillomycin D, iturin A and mycosubtilin operons, our results showed that intergenic module replacement have occurred between B. subtilis lipopeptide synthetases including the iturin family and the plipastatin and fengycin family.