Genome Sequences of 14 Escherichia coli O157:H7 Strains Isolated before and during the Time Frame of the 2018 Multistate Outbreak Associated with Romaine Lettuce.
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SUBMITTER: Stanton E
PROVIDER: S-EPMC7365793 | biostudies-literature | 2020 Jul
REPOSITORIES: biostudies-literature
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Genome Sequences of 14 Escherichia coli O157:H7 Strains Isolated before and during the Time Frame of the 2018 Multistate Outbreak Associated with Romaine Lettuce.
Stanton Eliot E Wagner Sara S Florek Kelsey R KR Kaspar Charles W CW
Microbiology resource announcements 20200716 29
Several outbreaks of <i>Escherichia coli</i> O157:H7 associated with contaminated leafy green vegetables have been documented. Here, we report the draft genome sequences of 14 strains isolated from human patients in the state of Wisconsin during a multistate outbreak in early 2018 that was linked to consumption of romaine lettuce. ...[more]
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Project description:BackgroundIn 2017, we conducted a multistate investigation to determine the source of an outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections, which occurred primarily in children.MethodsWe defined a case as infection with an outbreak strain of STEC O157:H7 with illness onset between January 1, 2017, and April 30, 2017. Case patients were interviewed to identify common exposures. Traceback and facility investigations were conducted; food samples were tested for STEC.ResultsWe identified 32 cases from 12 states. Twenty-six (81%) cases occurred in children <18 years old; 8 children developed hemolytic uremic syndrome. Twenty-five (78%) case patients ate the same brand of soy nut butter or attended facilities that served it. We identified 3 illness subclusters, including a child care center where person-to-person transmission may have occurred. Testing isolated an outbreak strain from 11 soy nut butter samples. Investigations identified violations of good manufacturing practices at the soy nut butter manufacturing facility with opportunities for product contamination, although the specific route of contamination was undetermined.ConclusionsThis investigation identified soy nut butter as the source of a multistate outbreak of STEC infections affecting mainly children. The ensuing recall of all soy nut butter products the facility manufactured, totaling >1.2 million lb, likely prevented additional illnesses. Prompt diagnosis of STEC infections and appropriate specimen collection aids in outbreak detection. Child care providers should follow appropriate hygiene practices to prevent secondary spread of enteric illness in child care settings. Firms should manufacture ready-to-eat foods in a manner that minimizes the risk of contamination.
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Project description:Field trials were conducted in July-August and October 2012 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, California. One-half milliliter of rabbit, chicken, or pig fecal slurry, containing an average of 4.05 × 107 CFU E. coli O157:H7 (C0), was inoculated onto the upper (adaxial) surface of a lower leaf on 288 heads of lettuce per trial immediately following a 2.5 h irrigation event. To estimate the bacterial inactivation rate as a function of time, fecal matrix, irrigation and seasonal climate effects, sets of lettuce heads (n = 28) were sampled each day over 10 days and the concentration of E. coli O157:H7 (Ct) determined. E. coli O157:H7 was detected on 100% of heads during the 10-day duration, with concentrations ranging from ≤340 MPN/head (∼5-log reduction) to >3.45 × 1012 MPN/head (∼5-log growth). Relative to C0, on day 10 (Ct = 12) we observed an overall 2.6-log and 3.2-log mean reduction of E. coli O157:H7 in July and October, respectively. However, we observed relative maximum concentrations due to bacterial growth on day 6 (maximum Ct = 8) apparently stimulated by foliar irrigation on day 5. From this maximum there was a mean 5.3-log and 5.1-log reduction by day 10 (Ct = 12) for the July and October trials, respectively. This study provides insight into the inactivation and growth kinetics of E. coli O157:H7 on romaine lettuce leaves under natural field conditions. This study provides evidence that harvesting within 24 h post irrigation has the potential to increase the concentration of E. coli O157:H7 contamination, if present on heads of romaine lettuce; foliar irrigation can temporarily stimulate substantial regrowth of E. coli O157:H7.
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Project description:Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that Escherichia coli O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol. 74:5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157.
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Project description:In the United States, due to the limited information about the microbial quality and safety of fresh produce after the labeled open dates, unnecessary discarding of fresh produce in good conditions and food loss have been caused. The aim of this study was to address this knowledge gap and evaluate the microbial quality of commercial chopped Romaine lettuce (RL) on the "Use By" dates (UBD) and 5 days after the "Use By" dates (UBD5). The microbial quality was evaluated using culture-dependent and culture-independent methods. Three brands of RL samples, from early and late harvest seasons, were purchased from local grocery stores and stored at 4°C until 5 days after their UBD. On the UBD and UBD5, bagged lettuce was opened, homogenized, diluted, and plated onto plate count agar and anaerobic agar to obtain total aerobic plate counts (APC) and total anaerobic plate counts (AnPC). For the culture-independent method, DNA was extracted from each sample homogenate and used for 16S rRNA gene sequencing. The culture-dependent results showed that there was no significant change in APC or AnPC between UBD and UBD5 samples. The APC and AnPC ranged from 5.71 ± 0.74 to 7.89 ± 0.10 Log CFU/g and 1.75 ± 0.08 to 7.32 ± 0.61 Log CFU/g, respectively. No significant difference in alpha diversity, based on observed features and Shannon index values, was detected between UBD and UBD5 samples using 16S rRNA sequencing. Similarly, no difference was observed in beta diversity based on the Jaccard distance matrixes and the weighted Unifrac distance matrixes. Taxonomic analysis revealed 128 genera in all RL samples. The top five genera were Pseudomonas (with relative abundance ranging from 16.47 to 92.72%), Serratia (0-52.35%), Weissella (0-42.42%), Pantoea (0.17-21.33%), and Lactococcus (0-24.30%). The differential abundance analysis based on the ANCOM test showed that no bacteria were detected to have significantly differential abundance in RL between UBD and UBD5. In summary, both the culture-dependent and culture-independent results showed that there was no significant difference in the microbial quality of RL before and shortly after the UBD.
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Project description:A regional, multistate investigation into a June-August 2013 cyclosporiasis outbreak was conducted in Nebraska, Iowa, and neighbouring states. Cases were confirmed on the basis of laboratory and clinical findings. Of 227 cases in Iowa (n = 140) and Nebraska (n = 87) residents, 162 (71%) reported dining at chain A/B restaurants - 96% reported house salad consumption. A case-control study identified chain A/B house salad as the most likely vehicle. Traceback was conducted to ascertain production lot codes of bagged salad mix (iceberg and romaine lettuce, red cabbage, and carrots) served as house salad in implicated restaurants. A single production lot code of salad mix supplied by both a common producer and distributor was linked to the majority of confirmed cases in persons reporting regional chain A/B exposure. The salad mix linked to illnesses contained imported romaine lettuce from two separate single-grower fields-of-origin and ⩾1 additional field from another grower.
| S-EPMC9150646 | biostudies-literature
Project description:An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.
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Project description: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.
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Project description:Romaine lettuce (Lactuca sativa) is an important staple of American agriculture. Unlike many vegetables, romaine lettuce is typically consumed raw. Phylloplane microbes occur naturally on plant leaves; consumption of uncooked leaves includes consumption of phylloplane microbes. Despite this fact, the microbes that naturally occur on produce such as romaine lettuce are for the most part uncharacterized. In this study, we conducted culture-based studies of the fungal romaine lettuce phylloplane community from organic and conventionally grown samples. In addition to an enumeration of all such microbes, we define and provide a discussion of the genera that form the "core" romaine lettuce mycobiome, which represent 85.5% of all obtained isolates: Alternaria, Aureobasidium, Cladosporium, Filobasidium, Naganishia, Papiliotrema, Rhodotorula, Sampaiozyma, Sporobolomyces, Symmetrospora and Vishniacozyma. We highlight the need for additional mycological expertise in that 23% of species in these core genera appear to be new to science and resolve some taxonomic issues we encountered during our work with new combinations for Aureobasidiumbupleuri and Curvibasidium nothofagi. Finally, our work lays the ground for future studies that seek to understand the effect these communities may have on preventing or facilitating establishment of exogenous microbes, such as food spoilage microbes and plant or human pathogens.
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