Ryan C. Fink

Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

ABSTRACT 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.

Enteric Pathogen-Plant Interactions: Molecular Connections Leading to Colonization and Growth and Implications for Food Safety

Leafy green vegetables have been identified as a source of foodborne illnesses worldwide over the past decade. Human enteric pathogens, such as Escherichia coli O157:H7 and Salmonella, have been implicated in numerous food poisoning outbreaks associated with the consumption of fresh produce. An understanding of the mechanisms responsible for the establishment of pathogenic bacteria in or on vegetable plants is critical for understanding and ameliorating this problem as well as ensuring the safety of our food supply. While previous studies have described the growth and survival of enteric pathogens in the environment and also the risk factors associated with the contamination of vegetables, the molecular events involved in the colonization of fresh produce by enteric pathogens are just beginning to be elucidated. This review summarizes recent findings on the interactions of several bacterial pathogens with leafy green vegetables. Changes in gene expression linked to the bacterial attachment and colonization of plant structures are discussed in light of their relevance to plant-microbe interactions. We propose a mechanism for the establishment and association of enteric pathogens with plants and discuss potential strategies to address the problem of foodborne illness linked to the consumption of leafy green vegetables.

Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane.

Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.

Presence and biological activity of antibiotics used in fuel ethanol and corn co-product production

Antibiotics are used in ethanol production to control bacteria from competing with yeast for nutrients during starch fermentation. However, there is no published scientific information on whether antibiotic residues are present in distillers grains (DG), co-products from ethanol production, or whether they retain their biological activity. Therefore, the objectives of this study were to quantify concentrations of various antibiotic residues in DG and determine whether residues were biologically active. Twenty distillers wet grains and 20 distillers dried grains samples were collected quarterly from 9 states and 43 ethanol plants in the United States. Samples were analyzed for DM, CP, NDF, crude fat, S, P, and pH to describe the nutritional characteristics of the samples evaluated. Samples were also analyzed for the presence of erythromycin, penicillin G, tetracycline, tylosin, and virginiamycin M1, using liquid chromatography and mass spectrometry. Additionally, virginiamycin residues were determined, using a U.S. Food and Drug Administration-approved bioassay method. Samples were extracted and further analyzed for biological activity by exposing the sample extracts to 10(4) to 10(7) CFU/mL concentrations of sentinel bacterial strains Escherichia coli ATCC 8739 and Listeria monocytogenes ATCC 19115. Extracts that inhibited bacterial growth were considered to have biological activity. Physiochemical characteristics varied among samples but were consistent with previous findings. Thirteen percent of all samples contained low (≤1.12 mg/kg) antibiotic concentrations. Only 1 sample extract inhibited growth of Escherichia coli at 10(4) CFU/mL, but this sample contained no detectable concentrations of antibiotic residues. No extracts inhibited Listeria monocytogenes growth. These data indicate that the likelihood of detectable concentrations of antibiotic residues in DG is low; and if detected, they are found in very low concentrations. The inhibition in only 1 DG sample by sentinel bacteria suggests that antibiotic residues in DG were inactivated during the production process or are present in sublethal concentrations.

Gene expression profiling of Escherichia coli in response to interactions with the lettuce rhizosphere

The objective of this study was to examine transcriptional changes in Escherichia coli when the bacterium was growing in the lettuce rhizoshpere.

Incidence of naturally internalized bacteria in lettuce leaves.

Lettuce is the fresh leafy vegetable most frequently involved in foodborne disease outbreaks. Human bacterial pathogens may be experimentally internalized into lettuce plants, but the occurrence of natural microflora inside lettuce leaves has not been elucidated. To characterize the endophytic microorganism residing in commercial lettuce leaves, two separate studies were conducted. First, a total of 30 and 25 heads of romaine and red leaf lettuce, respectively, served as the source of individual leaves which were surface sterilized, stomached, enriched in BHI broth for 24h and plated onto BHI agar for non-selective isolation of internalized microorganism. In a separate survey, 80 heads of each of the two types of lettuce were similarly processed, except that GN broth and MacConkey agar (MCA) were used for isolation of Gram negative bacteria. Thirty-eight out of 100 leaves were positive for internalized microorganisms, and Bacillus, Pseudomonas and Pantoea were the genera most frequently found in both types of lettuce. Members of the genus Erwinia were isolated from romaine lettuce only. In the second study, 21 and 60% of romaine and red leaf lettuce heads, respectively, had internalized bacteria capable of growing on MCA. Among the Gram negative strains, Pseudomonas and Pantoea genera were most frequently isolated. Enterobacter isolates were obtained from three red leaf samples. In summary, spore-forming bacteria and traditional epiphytic bacterial genera were frequently detected in surface-sterilized commercial lettuce leaves. Despite the common occurrence of internalized bacteria, only Enterobacter was related to Escherichia coli O157:H7 and Salmonella.

General response of Salmonella enterica serovar Typhimurium to desiccation: A new role for the virulence factors sopD and sseD in survival

Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella’s cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella’s survival during desiccation.

Transcriptional responses of Escherichia coli K-12 and O157

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Impact of distillers grain solids (DGS) and seasonality on the prevalence of Escherichia coli O157 at an abattoir in the U. S. Upper Midwest

ABSTRACT Enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 is carried asymptomatically by cattle gastrointestinal tract and the inclusion of distillers grains solids (DGS) in feed is thought to be a factor in the prevalence and persistence of EHEC O157 in a herd. The present study surveys the faecal prevalence of E. coli O157 in cattle processed at an abattoir in the Upper Midwest and its association with environmental factors and feeding practices. Faecal samples were collected from pre-processing cows during a 1-year period. E. coli O157 prevalence was estimated isolation of putative positives and confirmation of isolates by immunoassay and multiplex virulence genes PCR analysis. Overall, E. coli O157 was confirmed in 11.2% of samples. Prevalence during winter was the highest at 14% followed by summer (11.6%) and declined to less than 8% the rest of the year. Winter was the only season that had a statistically significant effect on prevalence. As a category unto itself, DGS feeding before arrival had no significant influence on faecal prevalence. However, we found a significant interaction of DGS feeding and summer. This observation is extremely relevant because it corroborates a previous study and suggests possible feeding practices to abate EHEC O157 presence during harvest.

Factors influencing the Salmonella internalization into seedpods and whole plants of Arachis hypogaea (L.)

Here we investigated whether Salmonella enterica serovar Typhimurium ATCC 14028 was capable of internalizing in peanut seedpods and plants when exposed to inoculated soil and the edaphic factors that influenced uptake. Intact dry Virginia (DV) and fresh green Virginia (GV) seedpods were exposed to soil containing 6.5 Log (CFU/g) Salmonella under different soil moisture conditions. Internalization of S. Typhimurium into peanut plants germinated in inoculated soil was also examined with and without Bradyrhizobium (Arachis) sp.NC92. Salmonella counts recovered from GV seedpods were on average of 2.0 Log (CFU/pod) less than those recovered from DV seedpods. The internalization in DV pods was only observed at soil water content of 15% or greater in a loamy sand soil. S. Typhimurium was detected inside peanut plant tissues during most testing times. Cells were recovered from stem samples (3.5 Log CFU/g) at greater levels than it was observed for root (2.6 Log CFU/g) and leaf (1.7 Log CFU/g) samples. Overall, recovery of Salmonella from stem, root, and leaf samples were lower when B. NC92 was inoculated on seeds before sowing, but this trend was not significant. Our observations suggest possible routes of contamination of Salmonella into peanut products from soil.